Note: Descriptions are shown in the official language in which they were submitted.
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SMART PEPTIDES AND TRANSFORMABLE NANOPARTICLES FOR CANCER
IMMUNOTHERAPY
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application Nos.
62/886,698 and
.. 62/886,718, both filed on August 14, 2019, each of which is incorporated
herein in its entirety
for all purposes.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER
FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
[0002] This invention was made with Government support under Grant Nos.
R01EB012569
and U01CA198880, awarded by the National Institutes of Health. The Government
has certain
rights in the invention.
BACKGROUND OF THE INVENTION
[0003] Clinical success in cancer immunotherapy in recent years has brought
great enthusiasm
to our war against cancer. Immune checkpoint receptor pathway blockade
monoclonal antibodies
such as anti-PD-1, anti-PD-L1, and anti-CTLA-4 can reverse T effector cell
(Teff) dysfunction
and exhaustion, resulting in dramatic tumour shrinkage and sometimes complete
remission in
some patients, even with late stage metastatic diseases. However, the response
rate varies greatly
between tumour types: up to 40% in melanoma, 25% in non-small cell lung
cancer, but <10% in
most other tumour types. To date, US Food and Drug Administration (FDA) had
approved seven
immune checkpoint blockade monoclonal antibodies (ICB-Ab): one CTLA-4
inhibitor
(ipilimumab), three PD-1 inhibitors (nivolumab, pembrolizumab, and
cemiplimab), and three
PD-Li inhibitors (atezolizumab, durvalumab, and avelumab), used either alone,
or in
combination with other chemotherapies, against a range of tumour types.
[0004] Tumour microenvironment (TME), comprised of immune and stromal cells,
vasculature, extracellular matrix, cytokines, chemokines, and growth factors,
can all influence
tumour response to immune checkpoint blockage (ICB) therapies. Emerging data
indicates that
defects in Teff cell homing to the tumour sites is a critical factor in
resistance to ICB therapy.
Other mechanisms of ICB resistance include the presence of immunosuppressive
regulatory T
cells (Tregs), myeloid-derived suppressor cells (MDSCs), and M2 macrophages at
the tumour
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sites. Elevated level of CCL5, CCL17, CCL22, CXCL8 and CXCL12 facilitates the
recruitment
of Tregs and MDSCs to the TME, resulting in a diminished ICB response. In
contrast, CXCL9
and CXCL10 promote homing of cytotoxic T-cells (CTLs) to the tumour sites,
boosting anti-
tumour immune response; transforming growth factor beta (TGF-13) does the
opposite and also
upregulates Tregs. VEGF upregulates inhibitory receptors on CTLs, contributing
to their
exhaustion. Upregulation of other immune checkpoint receptors such as mucin
domain-3 protein
(TIM-3), lymphocyte-activation gene 3 (LAG-3), B and T lymphocyte attenuator
(BTLA), T-cell
immunoreceptor, tyrosine-based inhibition motif domain (TIGIT), and V-domain
immunoglobulin-containing suppressor of T-cell activation (VISTA) has been
implicated in ICB
resistance. Co-expression of these checkpoint receptors can lead to T cell
exhaustion. Oncogenic
or tumour suppressor pathways, such as mitogen-activated protein kinase (MAPK)
and PI3K-y in
the cancer cells can also influence TME by altering the immune cell
compositions and cytokine
profile, contributing to ICB resistance. Inhibitors against these pathways
have been found to
improve ICB response.
[0005] In an attempt to overcome ICB resistance, many combination therapeutic
strategies
have been tried preclinically and clinically. These include the addition of
the following drugs to a
ICB-Ab: one other ICB-Ab (antibodies against CTLA-4, PD-1, PD-L1, LAG-3 and
TIM-3),
chemotherapeutic agents (paclitaxel, gemcitabine and carboplatin), radiation
therapy, targeted
therapy (inhibitors against PI3K, VEGF, BRAF/MEK, IDO, A2AR, FGFR, EGFR, PARP
and
.. mTOR), macrophage inhibitors (inhibitors against CSF1R and ARG1),
cytokine/chemokine
inhibitors (inhibitors against CXCR4, CXCR2 and TGF-13), epigenetic modulators
(histone
deacetylase inhibitors and hypomethylating agents), immunomodulatory agents
(antibodies
against 0X40, 41BB, GITR, CD40 and ICOS), adoptive cell transfer therapy (car
T, TIL and
TCR), and modulation of gut microbiome.
[0006] Advancement and optimization of nano-immunotherapy lie in the
development of
innovative approaches to enhance the specificity and controllability of
immunotherapeutic
interventions, targeting desired cell types at the TME. Advanced
bionanomaterials or approaches
in a more controlled manner could enhance immunotherapeutic potency by
increasing the
accumulation and prolonging the retention of immunomodulatory and immune cell
homing
agents at the TME while sparing the normal tissues and organs, thus reducing
off-target adverse
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effects such as systemic cytokine storm. In situ assembly of nanomaterial has
been demonstrated
to improve the performance of bioactive molecules. One plausible explanation
is that T cell
targeting ligands and/or immunomodulatory agents incorporated into in situ
fibrillar-
transformable nanoplatform, will generate nanofibrillar networks at the TME,
enhancing Teff
cells homing to the tumour sites and improving immunotherapeutic efficacy,
with or without
additional ICB therapy.
[0007] Human epidermal growth factor receptor 2 (HER2) is overexpressed in
over 20% breast
cancers, and to a lesser degree in gastric cancers, colorectal cancer, ovarian
cancers and bladder
cancers. Unlike those cancers caused by mutated or fusion oncogenes (e.g. EGFR
in lung cancers
and Bcr-Abl in chronic myelocytic leukemia) which respond well to monotherapy,
cancers with
HER2 overexpression often require drug combinations. It is because this latter
group of tumours
are driven by gene amplification and massive overexpression of HER2. HER2 is a
receptor
tyrosine kinase that is normally activated via induced dimerization with
itself or with its family
members EGFR, HER3 or HER4. In HER2 positive tumours, HER2s are massively
overexpressed and constitutively dimerized, leading to unrelenting activation
of down-stream
proliferation and survival pathways and malignant phenotype.
[0008] Because of the high expression level of HER2, trastuzumab and
pertuzumab, the two
anti-HER2 monoclonal antibodies are ineffective as monotherapy against these
tumours. They
need to be given in combinations with other HER2-targeted therapy,
chemotherapy or hormonal
therapy. Herein, some embodiments describe a novel HER2-mediated, peptide-
based, and non-
toxic transformative nano-agent that is highly efficacious as a monotherapy
against HER2+
breast cancer xenograft models. This receptor-mediated transformable
nanotherapy (RMTN) is
comprised of peptide with unique domains that allow self-assembly to form
micelles under
aqueous condition and transformation into nanofibrils at the tumour site,
where HER2 is
encountered. The resulting nanofibrillar network effectively suppresses HER2
dimerization and
downstream signaling, and facilitates tumour cell death.
[0009] Herein, smart supramoi ecitlar materials for cancer ilnimmotherapy were
constructed.
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BRIEF SUMMARY OF THE INVENTION
[0010] In one embodiment, the present invention provides a compound of formula
(I): A-B-C
(I), wherein A is a hydrophobic moiety; B is a peptide, wherein the peptide
forms a beta-sheet;
and C is a hydrophilic targeting ligand, wherein the hydrophilic targeting
ligand is a LLP2A
prodrug, LLP2A, LXY30, LXW64, DUPA, folate, a LHRH peptide, a HER2 ligand, an
EGFR
ligand, or a toll-like receptor agonist CpG oligonucleotides.
[0011] In another embodiment, the present invention provides a compound of
formula (I): A-
B-C (I), wherein A is a hydrophobic moiety; B is a peptide, wherein the
peptide forms a beta-
sheet; and C is a hydrophilic targeting ligand, wherein the hydrophilic
targeting ligand is a
.. LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, a LHRH peptide, a HER2 ligand, an
EGFR
ligand, or a toll-like receptor agonist CpG oligonucleotides and wherein when
the hydrophobic
moiety is bis-pyrene, then C is a LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, a
LHRH
peptide, an EGFR ligand, or a toll-like receptor agonist CpG oligonucleotides.
[0012] In another embodiment, the present invention provides a nanocarrier
having an interior
and an exterior, the nanocarrier comprising a plurality of compounds of the
present invention,
wherein each compound self-assembles in an aqueous solvent to form the
nanocarrier such that a
hydrophobic pocket is formed in the interior of the nanocarrier, and a
hydrophilic group self-
assembles on the exterior of the nanocarrier.
[0013] In another embodiment, the present invention provides a nanocarrier
having an interior
and an exterior, the nanocarrier comprising a plurality of a first conjugate
and a second conjugate
wherein the first conjugate comprises formula (I): A-B-C (I); and the second
conjugate
comprises formula (II): A'-B'-C' (II) wherein: A and A' are each independently
a hydrophobic
moiety; B and B' are each independently a peptide, wherein each peptide
independently forms a
beta-sheet; and C and C' are each independently a hydrophilic targeting
ligands, wherein each
hydrophilic targeting ligand is independently a LLP2A prodrug, LLP2A, LXY30,
LXW64,
DUPA, folate, a LHRH peptide, a HER2 ligand, an EGFR ligand, or a radiometal
chelator; and
wherein A and A' are different hydrophobic moieties and/or C and C' are
different hydrophilic
targeting ligands.
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[0014] In another embodiment, the present invention provides a method of
forming
nanofibrils, comprising contacting a nanocarrier of the present invention with
a cell surface or
acellular component at a tumor microenvironment, wherein the nanocarrier
undergoes in situ
transformation to form fibrillary structures, thereby forming the nanofibrils.
[0015] In another embodiment, the present invention provides a method of
treating a disease,
comprising administering to a subject in need thereof, a therapeutically
effective amount of a
nanocarrier of the present invention, wherein the nanocarrier forms
nanofibrils in situ after
binding to a cell surface or acellular component at the tumor
microenvironment, thereby treating
the disease.
[0016] in another embodiment, the present invention provides a method of
imaging,
comprising administering to a subject to be imaged, an effective amount of a
nanocarrier of the
present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIGs. 1A-1F show assembly and fibrillar-transformation of transformable
peptide
.. monomer 1 (TPM1') BP-FFVLK-YCDGFYACYMDV. FIGs. 1A-1B show changes in UV¨vis
absorption (FIG. 1A) and fluorescence (FIG. 1B)of NPs1 upon gradual addition
of water into
DMSO solution of NPs1 with the H20:DMS0 from 0:100, 20:80, 40:60, 60:40,
80:20, 90:10,
98:2 and 99.5:0.5. Ex = 380 nm. FIG. 1C TEM images of initial NPs1 and
nanofibers (NFs1)
transformed by NPs1 interaction with HER2 protein (Mw "--=, 72 KDa) at
different time points
(0.5, 6, 24 h). The scale bar in d: 100 nm. FIGs. 1D-1F- show variation of
size distribution
(FIG. 1D), CD spectra (FIG. 1E) and fluorescence signal (FIG. 1F) of initial
NPs1 and NFs1 at
the different time points. The molar ratio of HER2 peptide/HER2 protein was
approximately
1000:1.
[0018] FIGs. 2A-211 show the morphological characterizations of fibrillar-
transformable NPs1
co-culture with HER2 positive cancer cells. FIGs. 2A-2C show cellular
fluorescence distribution
images of NPs1 interaction with SKBR-3 cells (HER2+) (FIG. 2A), BT474 cells
(HER2+)
(FIG. 2B) and MCF-7 cells (HER2-) (FIG. 2C) at 6 h time point. Scale bar in
FIGs 2A-2C: 50
pm. FIG. 2D shows Western blot and quantitative analysis of relative HER2
protein expression
in MCF-7 cells and MCF-7/C6 cells. ***P < 0.001. FIG. 2E shows cellular
fluorescence
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distribution images of NPs1 interaction with MCF-7/C6 cells (HER2+) at the
different time
points (0.5, 6, 24 h). Scale bar in e: 50 gm. FIG. 2F shows fluorescence
binding distribution
images of the nanofibrillar network of NFs1 and HER2 antibody (29D8, rabbit,
different receptor
binding site with HER2 peptide of NPs1) on the cell membrane of MCF-7/C6
cells. HER2
antibody was used to label HER2 receptors. FIG. 2G shows SEM images of
untreated MCF-
7/C6 cells and cells treated by NPs1 for 6 h and 24 h. FIG. 211 shows TEM
images of untreated
MCF-7/C6 cells and cells treated by NPs1 for 24 h. The red arrow shows
fibrillar network. The
concentration of NPs1 was 50 pM.
[0019] FIGs 3A-3G show the extracellular and intracellular mechanisms of
fibrillar-
transformable NPs interaction with MCF-7/C6 breast cancer cells. FIG. 3A shows
cellular
fluorescence distribution images of NPs1, NPs2 and HER2 antibody (29D8,
rabbit, different
receptor binding site with HER2 peptide of NPs1 and NPs2) binding HER2
receptors of MCF-
7/C6 cells, respectively. HER2 antibody was used to label HER2 receptors. The
concentration of
NPs1 and NPs2 were 50 M. The scale bar in a: 20 gm. FIG. 3B shows the
viability of MCF-
7/C6 cells incubated with NPs1-4 at the different concentration (n = 3). *P
<0.05, **P <0.01.
FIG. 3C shows Western blot analysis of apoptosis related proteins and HER2
total protein in
MCF-7/C6 cells treated by NPs1 for 24 h with different concentration. FIGs. 3D-
3E show
Western blot analysis of inhibition and disaggregation mechanism of HER2
protein dimer in
MCF-7/C6 cells treated by NPs1 for 24 h with different concentration (FIG. 3D)
and at 50 gM
under different time point (FIG. 3E). FIG. 3F shows Western blot analysis of
inhibition
mechanism of proliferation protein in MCF-7/C6 cells treated by NPs1 at 50 pM
under different
time point and at 24 h under different concentration. FIG. 3G shows Western
blot analysis of
inhibition mechanism of proliferation protein in MCF-7/C6 cells treated by
NPs1-4 and
Herceptin (HP) at 36 h. The concentration of NPs1-4 were 50 gM, and the
concentration of
Herceptin was 15 gg/mL as a positive control group.
[0020] FIG. 4A-4F show in vivo evaluation of fibrillar-transformable NPs. FIG.
4A show
time-dependent ex vivo fluorescence images and FIG. 4B show quantitative
analysis of tumour
tissues and major organs (heart, liver, spleen, lung, kidney, intestine,
muscle and skin) collected
at 10, 24, 48, 72 and 168 h post-injection of NPs1. In FIG. 4B ***P < 0.001,
the fluorescence
signal in tumour tissue at 72 h and 168 h compared with other organs displays
tumour
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accumulation and in situ transformation of fibrillar network with long
retention time; ***P <
0.001, the fluorescence signal in liver and kidney at 10 h compared with that
at 72 and 168 h
displays that NPs1 could be removed rapidly from liver and kidney. FIG. 4C
shows the
fluorescence distribution images and H&E image of NPs1 in tumour tissue and
normal skin
tissue at 72 h post-injection (green color: BP of NPs1; blue color: DAPI;
scale bar in c: 100 pm).
FIG. 4D shows time-dependent ex vivo fluorescence images of tumour tissues and
major organs
collected at 72 h post-injection of NPs2-4. FIG. 4E shows quantitative
analysis of tumour tissues
and livers collected at 72 h post-injection of NPs1-4. In FIG. 4E ***P <0.001,
the fluorescence
signal of tumour tissue in NPs1 group compared with that in other control
groups displays that
fibrillar networks in NPs1 group promote long retention time in tumour site.
FIG. 4F shows
TEM images of distribution in tumour tissue and in situ fibrillar
transformation of NPs1-4 at 72 h
post-i.v. injection and untreated group. The dose of NPs1-4 were 8 mg/Kg per
injection. In FIG.
4F, "C" means MCF-7/C6 cell; "N" means cell nucleus.
[0021] FIG. 5A-5K show anti-tumour activity of NPs in Balb/c nude mice bearing
HER2
positive breast tumour. FIG. 5A shows schematic illustration of tumour
inoculation and
treatment protocol of mice. FIGs. 5B-5C show observation of the tumour
inhibition effect (FIG.
5B) and weight change of mice (FIG. 5C) in subcutaneous tumour model during
the 40 days of
treatment (n = 8 per group; the dose of NPs1-4 were 8 mg/Kg per injection).
**P < 0.01, ***P <
0.001. FIG. 5D shows cumulative survival of different treatment groups of mice
bearing MCF-
7/C6 breast tumours. FIG. 5E shows schematic illustration of three times
treatment protocol of
mice for tumour tissue analysis. FIG. 5F shows the fluorescence distribution
images in tumour
tissue and H&E anti-tumour image post three times injection of NPs1 (green
color: BP of NPs1;
blue color: DAPI; scale bar in f: 100 pm). FIG. 5G shows representative TEM
images of late
membrane rapture and cell death by the nanofibrillar network after injection
of NPs1 three times.
The red arrow shows fibrillar network. FIG. 511 shows Ki-67 stain images of
tumour tissues
treated by different groups after injection three times. Scale bar in h: 25
pm. FIG. 51 shows
Western blot analysis of inhibition mechanism of HER2 protein and
proliferation proteins in
MCF-7/C6 tumour tissues treated by different groups after injection three
times. FIGs. 5J-5K
shows oservation of the tumour inhibition effect in subcutaneous tumour SKBR-3
(FIG. 5J) and
BT474 HER2 positive breast cancer (FIG. 5K) models during the 40 days of
treatment (n = 8 per
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group; the dose of NPs1 were 8 mg/Kg per injection). ***P < 0.001 compared
with PBS control
group.
[0022] FIG. 6 shows chemical structure and mass spectra via MALDI-TOF of
transformable
peptide monomer 1 BP-FFVLK-YCDGFYACYMDV.
[0023] FIG. 7 shows chemical structure and mass spectra via MALDI-TOF of
transformable
peptide monomer 2 BP-GGAAK-YCDGFYACYMDV.
[0024] FIG. 8 shows chemical structure and mass spectra via MALDI-TOF of
transformable
peptide monomer 3 BP-FFVLK-PEG.
[0025] FIG. 9 shows chemical structure and mass spectra via MALDI-TOF of
transformable
peptide monomer 4 BP-GGAAK-PEG.
[0026] FIG. 10 shows effect of HER2 protein/peptide ligand ratio on fibrillar
transformation.
TEM images and particle size measurements of NPs1 were obtained after
incubation with
soluble HER2 protein for 24 h in PBS solution. NPs1 concentration was
maintained constant at
M. The scale bar is 200 nm. The HER2 protein/peptide ligand ratio is labeled
for each
15 micrograph. Experiments were repeated three times.
[0027] FIG. 11A shows bbservation on the anti-tumour effect in subcutaneous
SKBR-3
tumour during the 40 days of treatment (n = 6 per group; the dose of NPs1-4
were 8 mg/kg per
injection, q.o.d.; data are presented as the mean s.d.). The statistical
significance was
calculated via one-way ANOVA with a Tukey post-hoc test. *P< 0.05. FIGs. 11B-
11C show
20 body weight of mice bearing subcutaneous BT474 tumour (FIG. 11B) and
SKBR-3 tumour
(FIG. 11C) during the 40 days of treatment (n = 6 per group; data are
presented as the mean
s.d.). Red arrows depict each single i.v. injection.
[0028] FIG. 12 shows nanofibrillar networks promote T cell homing and
reprogram tumour
microenvironment for enhanced immunotherapy. Schematic illustration of self-
assembly and
fibrillar transformation of TPMs, and (I), (II), (III) process in tumour
tissue: in situ fibrillar
transformation of NPs, LLP2A conversion from proLLP2A, followed attracting and
targeting T
cells, and TAMs re-education of from M2 to M1 phenotype. TPMs, NPs, NFs, Ml-
TAM and
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M2-TAM represent transformable peptide monomers, nanoparticles, nanofibrils,
Ml-like
tumour-associated microphage and M2-like tumour-associated microphage,
respectively.
[0029] FIG. 13A-131I shows assembly and fibrillar transformation of
transformable peptide
TPM1 (LXY30-KLVFFK(Pa)) and TPM2 (proLLP2A-KLVFFK(R848)). FIG. 13A shows
schematic illustration of molecular structure and function of TPM1 and TPM2.
FIG. 13B shows
changes in fluorescence (FL) of T-NPs following the gradual addition of water
(from 0 to 99%)
to a solution of T-NPs in DMSO comprised of TPM1 and TPM2 at a 1:1 ratio;
excitation
wavelength, 405 nm. FIG. 13C shows TEM images of initial T-NPs and T-NPs
transformed into
nanofibrils (T-NFs) after interaction with soluble a3(31 integrin protein for
24 h (H20 to DMSO
ratio of 99:1). The concentration of T-NPs used in the experiment was 20 M.
The scale bars in c
are 100 nm. FIG. 13D shows variation in fluorescence signal of Pa in the
fibrillar-transformation
process of T-NPs to T-NFs over time. FIG. 13A Eshows TEM images of initial T-
NPs and T-
NFs after interaction with esterase, soluble a431 integrin protein or a431
integrin protein plus
esterase for 24 h (H20 to DMSO ratio of 99:1). The concentration of T-NPs used
in the
experiment was 20 M. The scale bars in e are 100 nm. FIGs. 13F-3G show
variation in size
distribution (FIG. 13F) and circular dichroism spectra (FIG. 13G) of initial T-
NPs and T-NFs
under different conditions. FIG. 1311 shows Tte in vitro release profile of
R848 from T-NFs over
time. The molar ratio of a3(31 or a431 integrin protein to peptide ligand was
approximately
1:1000. a.u., arbitrary units; mdeg, millidegrees.
[0030] FIG. 14 shows DLS experiment to confirm transformation of T-NPs to T-
NFs.The
peak at 20 nm gradually went down in the solution, while the peak around 700
nm went up.
[0031] FIG. 15A-151I shows morphological characterization of fibrillar-
transformable
nanoparticles after incubation with 4T1 murine breast cancer cells. FIG. 15A
shows cellular
fluorescence distribution images of T-NPs and UT-NPs interaction for 6 h with
4T1 cells. Scale
bar is 10 pin. Experiments were repeated three times. FIG. 15B shows cellular
fluorescence
signal retention images of 4T1 cells after exposure to T-NPs and UT-NPs for 6
h followed by
incubation with fresh medium without NPs for 18 h. Scale bar is 10 pin.
Experiments were
repeated three times. FIG. 15C shows representative TEM images of 4T1 cells
treated with T-
NPs and UT-NPs for 24 h, showing abundance of nanofibrils around cells treated
with T-NPs.
Scale bar is 200 nm. Experiments were repeated three times. The concentration
of T-NPs was
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50 M. FIG. 15D shows cellular fluorescence distribution images of Jurkat T-
lymphoma cells
(GFP labeled) after incubation with esterase-treated T-NPs. Jurkat cells were
used to mimic T-
lymphocytes, which also express a431 integrin. Scale bar is 10 um. Experiments
were repeated
three times. FIG. 15E shows representative SEM images of untreated 4T1 and
Jurkat cells, and
cells treated with T-NPs for 6 h. Scale bar is 10 um. Experiments were
repeated three times.
FIG. 15F shows experimental scheme and cellular fluorescence distribution
images of T-NPs
(fluorescent red), after interaction with 4T1 and GFP-labled Jurkat cells. It
shows nanofibrillar
networks covering 4T1 cells, which in turn could attract and bind Jurkat
malignant T-cells. Scale
bar is 10 um. Experiments were repeated three times. FIG. 5G shows
representative SEM
images of 4T1 and Jurkat cells after treatment with T-NPs (see FIG. 15F).
Experiments were
repeated three times. FIG. 1511 shows representative images of M2-like murine
macrophages
and subsequent re-education by T-NFs, T-NFs plus esterase, or R848 at
different time points.
Scale bar is 20 um. Experiments were repeated three times. Statistical
significance was
calculated using a two-sided unpaired t test; *P < 0.05, **P < 0.01, ***P <
0.001.
[0032] FIG. 16A-16M shows in vivo evaluation of fibrillar-transformable
nanoparticles. FIG.
16A-16B show time-dependent ex vivo fluorescence (FL) images (FIG. 16A) and
quantitative
analysis (FIG. 16B) of tumour tissues and major organs (heart (H), liver (Li),
spleen (Sp), lung
(Lu), kidney (K), intestine (I), muscle (M) and skin (Sk)) collected at 10,
24, 48, 72, 120 and
168 h post-injection of T-NPs. Data are presented as mean s.d., n= 3
independent experiments.
FIG. 16C shows time-dependent ex vivo fluorescence (FL) images of tumour
tissues collected at
10, 24, 48, 72, 120 and 168 h post-injection of UT-NPs. Data are presented as
mean s.d., n= 3
independent experiments. FIG. 16D shows fluorescence (FL) quantification of
tumour tissues
collected at 10, 24, 48, 72, 120 and 168 h post-injection of T-NPs and UT-NPs.
FIG. 16E shows
representative TEM images of distribution in tumour tissue and in situ
fibrillar transformation of
T-NPs, UT-NPs and untreated control group at 72 h post-injection. "N" depicts
nucleus. FIG.
16F shows fluorescence (FL) distribution images of T-NPs in tumour tissue and
normal skin
tissue at 72 h post-injection (red, Pa of T-NPs; blue, DAPI; scale bars, 50
um). FIG. 16G shows
R484 distribution retention in tumour tissues at different time points post
injection of T-NPs and
UT-NPs. Dose of R848: 0.94 mg kg-1; data were mean s.d., n = 3 for each time
point. FIG.
1611 shows the expression of CXCL10 chemokine within the tumour tissues after
3 days of T-
NPs, UT-NPs and saline treatment (n = 3; data were mean s.d.). FIG. 16I-16K
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representative flow cytometric analysis images of CD45+CD3+ (FIG. 161),
CD8+/CD4+ (FIG.
16J) and CD4+Foxp3+ (FIG. 16K) T cell within the 4T1 tumours excised from mice
treated with
T-NPs, UT-NPs or saline control. FIG. 16L shows immunohistochemistry (IHC) of
tumours
excised from mice after treatment with T-NPs or UT-NPs. Representative images
are shown for
the IHC staining of T cells (CD8+, CD4+, Foxp3+) and macrophage markers (CD68,
CD163).
Scale bar is 100 pm. FIG. 16M shows the expression levels (qPCR assay) of IFN-
y, TGF-13,
IL12, IL10, Nos2 and Arg-1 in 4T1 tumours excised from mice 15 days after
treatment with T-
NPs or UT-NPs (n = 3; data were mean s.d.). Statistical significance was
calculated using a
two-sided unpaired t test; *P < 0.05, **P < 0.01, ***P < 0.001.
[0033] FIG. 17A-17G shows anti-tumour efficacy of fibrillar-transformable
nanoparticles in
Balb/c mice bearing 4T1 breast tumour. FIG. 17A shows experimental design:
orthotopic
tumour inoculation and treatment protocol; regimen 6 is T-NPs with all the 4
critical
components. FIG. 17B-17C show Oservation of tumour inhibitory effect (FIG.
17B) and weight
change (FIG. 17C) of mice bearing orthotopic 4T1 tumour over 21 d after
initiation of treatment
.. (n = 8 per group). Data are presented as mean s.d. FIG. 17D shows
cumulative survival of
different treatment groups of mice bearing 4T1 breast tumours. FIG. 17E shows
representative
flow cytometric analysis images of CD3+CD8+ T cell within the 4T1 tumours
excised from
treated mice on day 21. FIG. 17F shows H&E and IHC images of excised tumors.
Representative images are shown for the IHC staining of Ki67, T cells (CD8,
Foxp3) and
macrophage markers (CD68, CD163). Scale bar is 100 pm. FIG. 17G shows yhe
expression
levels (analyzed by qPCR) of IFN-y, TNF-a, IL12, IL6, TGF-13, IL10, Nos2 and
Arg-1 in 4T1
tumours excised from mice on day 21 (data were mean s.d.). Statistical
significance was
calculated using a two-sided unpaired t test; *P < 0.05, **P < 0.01, ***P <
0.001.
[0034] FIG. 18A-18L shows anti-tumour efficacy of fibrillar-transformable
nanoparticles plus
anti-PD-1 therapy in mice bearing 4T1 breast tumour or Lewis lung tumour. FIG.
18A shows
experimental design: orthotopic tumour inoculation and treatment protocol (4
treatment arms;
regimen 4, 5 and 6 are the same as those shown in Fig. 4a). FIG. 18B shows
tumor response in
mice bearing orthotopic 4T1 tumour over 21 d of treatment (n = 8 per group).
Data are presented
as mean s.d. ***P < 0.001. FIG. 18C shows cumulative survival of the four
treatment groups.
FIG. 18D shows experimental design: Mice previously treated with T-NPs
(regimen 6) plus anti-
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PD-1 Ab were rechallenged with re-inoculation of cancer cells on day 90,
followed by three
q.o.d i.p. doses of anti-PD-1 Ab. FIG. 18E shows no anti-tumor immune memory
effect was
observed in same age naive mice. FIG. 18F shows anti-tumor immune memory
effect was
observed in mice previously treated with T-NPs and anti-PD-1 Ab. FIG. 18G
shows cumulative
survival of naive mice and previously T-NPs plus anti-PD-1 treated mice. FIG.
1811-181 show
IFN-y (FIG. 1811) and TNF-a (FIG. 181) level in mouse sera 6 days after mice
were
rechallenged with 4T1 tumor cells and a day after the last dose of anti-PD-1
Ab. FIG. 18J-18K
shows Observation of tumour inhibitory effect (FIG. 18J) and weight change
(FIG. 18K) of
mice bearing subcutaneous murine Lewis lung tumour over 21 d after initiation
of treatment
(n = 8 per group); Treatment protocol followed experiment design in FIG. 18A,
5 cycles (i.v.
regimen 4-6 and i.p. anti-PD-1. Data are presented as mean s.d. FIG. 18L
shows cumulative
survival of different treatment groups of mice bearing murine Lewis lung
tumours. Statistical
significance was calculated using a two-sided unpaired t test; *P < 0.05, **P
< 0.01,
***P < 0.001.
[0035] FIG. 19A shows structure of CPTNPs (BP-k-1-v-f-f-k-(08) where Green ¨
Bispyrene.
Blue ¨ hydrophobic bonding motif Red ¨ Cell-penetrating peptide. FIG. 19B
shows GG-
CPTNP (BP-k-1-v-g-g-k-(08) with similar coloration to A where the duel
phenylalanine motif is
replaced with a duel glycine motif FIG. 19C shows DLS of CPTNPs (FF) and GG-
CPTNPs
(GG) in various pH. FIG. 19D shows fluorescence of CPTNP nanoparticles and
CPTNP
monomers where the AIEE effect of BP may be observed. FIG. 19E shows Zeta
potential of FF
and GG CPTNPs measured at 50p.M. (a:b, p< 0.0005) FIG. 19F shows TEM images of
CPTNPs in various specified environments. Scale bar is 1001am in each image.
[0036] FIG. 20 shows Chemical structure and mass spectra via MALDI-TOF of
transformable
peptide monomer (TPM) 1 LXY30- KLVFFK(Pa), 2 proLLP2A-KLVFFK(R848), 3 LXY30-
KAAGGK(Pa), 4 proLLP2A-KAAGGK(R848). Experiments were repeated three times.
[0037] FIG. 21A shows TEM images and size distribution of NPsTPM1, NPsTPM1 and
T-
NPs at the H20 and DMSO ratio of 99:1. Experiments were repeated three times.
FIG. 21B
shows the critical aggregation concentration (CAC) of T-NPs was measured by
using pyrene as a
probe. Experiments were repeated three times. FIG. 21C shows nanoparticle
stability of T-NPs
in serum and protease (PBS solution of pH 7.4 with/without 10% FBS and
protease) at 37 oC
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was measured by dynamic light scattering. Data are presented as the mean
s.d., n = 3
independent experiments. FIG. 21D shows TEM images of freshly prepared T-NPs
and T-NPs
after 24 h in PBS solution. Experiments were repeated three times. FIG. 21E
show Tte CAC of
T-NFs was measured by using pyrene as a probe. Experiments were repeated three
times. The
scale bar in all TEM images is 100 nm. The concentration of T-NPs used in FIG.
21A, 21C, and
21D was 20 M.
[0038] FIG. 22 shows TEM images of initial UT-NPs and UT-NPs interaction with
a3(31
integrin protein for 24 h. The molar ratio of (13(31 integrin protein/peptide
ligand was
approximately 1:1000. The scale bar is 100 nm. The concentration used in the
experiment was 20
M. Experiments were repeated three times.
[0039] FIG. 23 shows biotinylated LXY30 peptide (blue curve) and negative
control (red
curve) incubation with 4T1 cells were analyzed with flow cytometry.
Experiments were repeated
three times. 3x105 cells incubated with 1 pM biotinylated LXY30 for 30 mm on
ice, after
washing with PBS followed by incubation with 1:500 streptavidin-PE (1mg/mL)
for 30min, then
run with flow cytometry.
[0040] FIG. 24 shows viability of 4T1 cells after incubation with T-NPs and UT-
NPs at
different concentrations for 48 h. Data are presented as mean s.d., n = 3
independent
experiments.
[0041] FIG. 25 shows blood test parameters in terms of red blood cells (RBC),
white blood
cells (WBC), platelets, hemoglobin, lymphocyte and total protein of healthy
Balb/c mice, after 8
q.o.d. intravenous injections of T-NPs and UT-NPs (13 mg/kg per injection).
Data are presented
as the mean s.d., n = 3 independent experiments.
[0042] FIG. 26 shows blood test parameters in terms of liver function
creatinine, alanine
transaminase, aspartate transaminase, albumin, alkaline phosphatase, total
bilirubin of healthy
Balb/c mice after 8 q.o.d. intravenous injection of T-NPs and UT-NPs (13 mg/kg
per injection).
Data are presented as the mean s.d., n = 3 independent experiments.
[0043] FIG. 27 shows in vivo blood pharmacokinetics and parameter of T-NPs and
UT-NPs
(Data are presented as the mean s.d., n = 3 independent experiments). The C-
max, AUC and
T1/2 (hours) were calculated by Kinetica 5Ø
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DETAILED DESCRIPTION OF THE INVENTION
I. GENERAL
[0044] The present invention provides compounds comprising a hydrophobic
moiety, a beta-
sheet peptide, and a hydrophilic targeting ligand, which can form
nanocarriers. The nanocarriers
can comprise a plurality of one conjugate or two different conjugates. The
nanocarriers can
transform in situ to form nanofibrils for treatment of diseases and imaging.
II. DEFINITIONS
[0045] Unless specifically indicated otherwise, all technical and scientific
terms used herein
have the same meaning as commonly understood by those of ordinary skill in the
art to which
this invention belongs. In addition, any method or material similar or
equivalent to a method or
material described herein can be used in the practice of the present
invention. For purposes of the
present invention, the following terms are defined.
[0046] "A," "an," or "the" as used herein not only include aspects with one
member, but also
include aspects with more than one member. For instance, the singular forms
"a," "an," and
"the" include plural referents unless the context clearly dictates otherwise.
Thus, for example,
reference to "a cell" includes a plurality of such cells and reference to "the
agent" includes
reference to one or more agents known to those skilled in the art, and so
forth.
[0047] "Hydrophobic moiety" refers to the part of the compound which is
substantially
insoluble in water. For example, when a plurality of compounds are present
which comprise a
hydrophobic and hydrophilic moiety, the hydrophobic moiety will orient
themselves in such a
way as to avoid and minimize interaction with water molecules. Hydrophobicity
of a moiety can
be determined by one of ordinary skill in the art by using the octanol-water
reference system to
measure the logarithm of the partition coefficient (logP value). LogP values
greater than 0
indicate the compound is hydrophobic, with greater values indicating greater
hydrophobicity.
[0048] "Peptide" refers to a compound comprising two or more amino acids
covalently linked
by peptide bonds. As used herein, the term includes amino acid chains of any
length, including
full-length proteins.
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[0049] "Beta-sheet", also known as beta-pleated sheet, refers to the secondary
structure in
proteins and comprises beta strands stabilized by hydrogen bonds. Beta-strands
can stack parallel
or antiparallel to each other to form beta-sheets.
[0050] "Beta-sheet peptide domain" refers to a domain within a protein
structure comprising
beta-sheets.
[0051] "Beta-amyloid peptide" refers to peptides that form amyloid plaques in
the brain. The
formation of amyloid plaques in the brain is found in subjects with
Alzheimer's disease.
[0052] "Hydrophilic targeting ligand" refers to a portion of the compound that
can target cell
surface receptors, cell surface proteins, or extracellular components and are
hydrophilic.
Hydrophilicity can be determined by measuring the logP value of a compound,
wherein values
less than 0 indicate hydrophilicity. Lower values indicate higher
hydrophilicity. Targeting
ligands can be used to target transmembrane receptors such as, but not limited
to integrins and
epidermal growth factor receptors, to delivery compounds, drugs, or components
of interest to
the cell or extracellular environment. Hydrophilic targeting ligands can
include, but are not
limited to peptides.
[0053] "Prodrug" refers to a compound that is biologically inactive, which
becomes
biologically active after being metabolized in situ. The prodrug can be
metabolized by
spontaneous reactions or enzymes within a mammal, resulting in an active
compound. Functional
groups useful in prodrugs include, but are not limited to esters, amides,
carbamates, oximes,
imines, ethers, phosphates, or beta-amino-ketones.
[0054] "LLP2A", "LXY30", and "LXW64" refer to compounds that can bind to an
integrin
protein. The structures of the three individual compounds are known by one of
skill in the art.
[0055] "DUPA" refers to a glutamate urea compound and can be used to deliver
cytotoxic
drugs to prostate cancer cells. DUPA, 243-(1,3-
dicarboxypropypureido]pentanedioic acid, has
the following structure:
0 0
H H
N.L
HO) IIN OH
....,:z... 0 ...;..->,
HO 0 0 OH .
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[0056] "LHRH peptide" refers to luteinizing hormone releasing hormone peptide,
and is
commercially available. LHRH peptide can be used to target ovarian and
prostate cancer cells.
[0057] "HER2 ligand" refers to a ligand that can bind to the HER2 protein.
Examples include,
but are not limited to anti-HER2 monoclonal antibodies, such as, but not
limited to trastuzumab
and pertuzumab and the EGFR ligands listed below.
[0058] "EGFR ligand" refers to a ligand that can bind to the EGFR protein.
Examples include,
but are not limited to EGF, TGF-alpha, HB-EGF, amhiregulin, betacellulin,
epigen, epiregulin,
neuregulin 1, neuregulin 2, neuregulin 3, and neuregulin 4.
[0059] "Toll-like receptor agonist" refers to a compound that binds to the
toll-like receptor on
.. cells, which plays a key role in the immune system. Binding to the receptor
can activate the
receptor to produce a biological response. An example of a toll-like receptor
agonist includes,
but is not limited to CpG oligonucleotides.
[0060] "CpG oligonucleotides", also known as CpG ODN, refer to cytosine-
guanosine
dinucleotide motifs. The two nucleotides can be linked by a phosphodiester
linker, or a modified
phosphorothioate linker.
[0061] "Dye" or "fluorescent dye" refers to a chemical molecule which emits
lights,
commonly in the 300-700 nm range, after excitation of the chemical molecule.
Upon absorption
of transferred light energy (e.g., photon), a dye molecule goes into an
excited state. As the
molecule exits the excited state, it emits the light energy in the form of
lower energy photon
(e.g., emits fluorescence) and returns the dye molecule to its ground state. A
dye can be a natural
chemical compound or a synthetic chemical compound. Dyes include, but are not
limited to
cyanines, porphyrins, and bis-pyrenes.
[0062] "Porphyrin" refers to any compound, with the following porphin core:
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wherein the porphin core can be substituted or unsubstituted.
[0063] "Bis-pyrene" refers to a compound which comprises two pyrene subunits
covalently
linked to each other. The two pyrene subunits can be linked directly or
through a linker. The
linker can be any linker known to one of skill in the art, such as but not
limited to, alkylenes,
alkenylenes, alkynylenes, aryls, heteroaryls, aryl ketones, ketones, amines,
amides, and ureas,
wherein the linker can be substituted.
[0064] "Radiometal chelator" refers to a polydentate ligand binding to a
single central metal
atom or ion. The metal atom or ion can be a radioactive isotope of the metal.
Radiometal
chelators include, but are not limited to Gd(III) chelators, DOTA chelator and
NOTA chelator.
Gd(III) chelators include, but are not limited to gadopentetic acid, gadoteric
acid, gadodiamide,
gadobenic acid, gadoteridol, gadoversetamide, and gadobutrol.
[0065] "Cyanine" or "cyanine dye" refers to a synthetic dye family belonging
to a polymethine
group. Cyanines can be used as fluorescent dyes for biomedical imaging.
Cyanines can be
streptocyanines (also known as open chain cyanines), hemicyanines, and closed
chain cyanines.
Closed chain cyanines have nitrogens which are each independently part of a
heteroaromatic
moiety.
[0066] "Drug" refers to an agent capable of treating and/or ameliorating a
condition or disease.
A drug may be a hydrophobic drug, which is any drug that repels water.
Hydrophobic drugs
useful in the present invention include, but are not limited to, deoxycholic
acid, taxanes,
doxorubicin, etoposide, irinotecan, SN-38, cyclosporin A, podophyllotoxin,
Carmustine,
Amphotericin, Ixabepilone, Patupilone (epothelone class), rapamycin and
platinum drugs. Other
drugs includes non-steroidal anti-inflammatory drugs, and vinca alkaloids such
as vinblastine
and vincristine. The drugs of the present invention also include prodrug
forms. One of skill in
the art will appreciate that other drugs are useful in the present invention.
[0067] "Chemotherapeutic agent" refers to chemical drugs that can be used in
the treatment of
diseases such as, but not limited to, cancers, tumors and neoplasms. In some
embodiments, a
chemotherapeutic agent can be in the form of a prodrug which can be activated
to a cytotoxic
form. Chemotherapeutic agents commonly known by one of ordinary skill in the
art can be used
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in the present invention. Chemotherapeutic agents include, but are not limited
to resiquimod,
gardiquimod, and imiquimod.
[0068] "Immunomodulatory agent" refers to a type of drug which can modify
immune
responses by stimulating or suppressing the immune system. Immunomodulatory
agents include,
but are not limited to resiquimod, gardiquimod, and imiquimod.
[0069] "Anti-HER2 rhumAb 4D5" refers to a type of HER2 antibody, and is also
known as
trastuzumab. Trastuzumab is commonly used to treat breast and stomach cancer
and is
commercially available. Trastuzumab comprises at least 50% peptide sequence
identity of SEQ
ID NO: 4. The peptide sequence of trastuzumab is described in "Rationally
designed anti-
HER2/neu peptide mimetic disables P185HER2/neu tyrosine kinases in vitro and
in vivo" (Park
et al. Nat Biotechnol. 2000 Feb;18(2):194-8.)
[0070] "CDR-H3 loop" refers to a region inside a HER2 antibody involved with
antigen
binding.
[0071] "Nanocarrier" or "nanoparticle" refers to a micelle resulting from
aggregation of the
compounds of the invention. The nanocarrier of the present invention can have
a hydrophobic
core and a hydrophilic exterior.
[0072] "Nanofibrils" refer to tubular, rod-like fibrils which have a diameter
ranging from tens
to hundreds of nanometers. Nanofibrils can have high length-to-diameter
ratios. Nanofibrils of
the present invention can be formed by an in situ transformation of the
nanoparticles after
binding at the targeted site.
[0073] "Fibrillary structures" refer to linear, rod-like fibrils with
diameters on the order of
nanometers to micrometers and have a high length-to-diameter ratio. Fibrillary
structures may
include biopolymers. Fibrillary structures include, but are not limited to,
nanofibrils and
microfibrils.
[0074] "Cell surface" refers to the plasma membrane, which separates the
extracellular space
from the interior of the cell. The cell surface comprises the lipid bilayer,
proteins, and
carbohydrates.
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[0075] "Acellular component" refers to the extracellular environment of a cell
and includes,
but is not limited to the extracellular matrix, extracellular vesicles, and
cytokines surround a cell.
The extracellular matrix comprises collagens, fibronectin, and other matrix
proteins. Ligands and
compounds can interact with an acellular component of cancerous cells to
affect the growth of
cancer cells.
[0076] "Tumor microenvironment" refers to tumor cells and the acellular
environment
surrounding it, including, but not limited to the extracellular matrix,
signaling molecules,
immune cells, stromal cells, vasculature, blood vessels, cytokines,
chemokines, growth factors,
and fibroblasts. Tumors can interact with the surround cells in the
microenvironment through the
lymphatic and circulatory systems to affect the growth and evolution of cancer
cells.
[0077] "Treat", "treating" and "treatment" refers to any indicia of success in
the treatment or
amelioration of an injury, pathology, condition, or symptom (e.g., pain),
including any objective
or subjective parameter such as abatement; remission; diminishing of symptoms
or making the
symptom, injury, pathology or condition more tolerable to the patient;
decreasing the frequency
or duration of the symptom or condition; or, in some situations, preventing
the onset of the
symptom. The treatment or amelioration of symptoms can be based on any
objective or
subjective parameter; including, e.g., the result of a physical examination.
[0078] "Administering" refers to oral administration, administration as a
suppository, topical
contact, parenteral, intravenous, intraperitoneal, intramuscular,
intralesional, intranasal or
subcutaneous administration, intrathecal administration, or the implantation
of a slow-release
device e.g., a mini-osmotic pump, to the subject.
[0079] "Subject" refers to animals such as mammals, including, but not limited
to, primates
(e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice
and the like. In certain
embodiments, the subject is a human.
[0080] "Therapeutically effective amount" or "therapeutically sufficient
amount" or "effective
or sufficient amount" refers to a dose that produces therapeutic effects for
which it is
administered. The exact dose will depend on the purpose of the treatment, and
will be
ascertainable by one skilled in the art using known techniques (see, e.g.,
Lieberman,
Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and
Technology of
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Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and
Remington: The
Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed.,
Lippincott, Williams &
Wilkins). In sensitized cells, the therapeutically effective dose can often be
lower than the
conventional therapeutically effective dose for non-sensitized cells.
[0081] "Cancer" refers to diseases with abnormal cell growth and divides
without
control. Cancer cells can spread locally or through the bloodstream and
lymphatic system to
other parts of the body. The term is also intended to include any disease of
an organ or tissue
characterized by poorly controlled or uncontrolled multiplication of normal or
abnormal cells in
that tissue and its effect on the body as a whole.
[0082] "Imaging" refers to using a device outside of the subject to determine
the location of an
imaging agent, such as a compound of the present invention. Examples of
imaging tools include,
but are not limited to, fluorescence microscopy, positron emission tomography
(PET), magnetic
resonance imaging (MRI), ultrasound, single photon emission computed
tomography (SPECT)
and x-ray computed tomography (CT). The positron emission tomography detects
radiation
from the emission of positrons by an imaging agent.
III. COMPOUNDS
[0083] In some embodiments, the present invention provides a compound of
formula (I): A-B-
C (I), wherein A is a hydrophobic moiety; B is a peptide, wherein the peptide
forms a beta-sheet;
and C is a hydrophilic targeting ligand. The hydrophilic targeting ligand can
include a HER2
ligand, and any other suitable target ligand.
[0084] In some embodiments, the present invention provides a compound of
formula (I): A-B-
C (I), wherein A is a hydrophobic moiety; B is a peptide, wherein the peptide
forms a beta-sheet;
and C is a hydrophilic targeting ligand, wherein the hydrophilic targeting
ligand is a LLP2A
prodrug, LLP2A, LXY30, LXW64, DUPA, folate, a LHRH peptide, a HER2 ligand, an
EGFR
ligand, or a toll-like receptor agonist CpG oligonucleotides.
[0085] In some embodiments, the present invention provides a compound of
formula (I)
wherein A is bis-pyrene; B is a peptide, wherein the peptide forms a beta-
sheet; and C is a HER2
ligand.
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[0086] In some embodiments, the present invention provides a compound of
formula (I): A-B-
C (I), wherein A is a hydrophobic moiety; B is a peptide, wherein the peptide
forms a beta-sheet;
and C is a hydrophilic targeting ligand, wherein when the hydrophobic moiety
is bis-pyrene, then
C is other than a HER2 ligand.
[0087] In some embodiments, the present invention provides a compound of
formula (I): A-B-
C (I), wherein A is a hydrophobic moiety; B is a peptide, wherein the peptide
forms a beta-sheet;
and C is a hydrophilic targeting ligand, wherein the hydrophilic targeting
ligand is a LLP2A
prodrug, LLP2A, LXY30, LXW64, DUPA, folate, a LHRH peptide, a HER2 ligand, an
EGFR
ligand, or a toll-like receptor agonist CpG oligonucleotides and wherein when
the hydrophobic
moiety is bis-pyrene, then C is a LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA,
folate, a
LHRH peptide, an EGFR ligand, or a toll-like receptor agonist CpG
oligonucleotides.
[0088] In some embodiments, the present invention provides a compound of
formula (I): A-B-
C (I), wherein A is a hydrophobic moiety; B is a peptide, wherein the peptide
forms a beta-sheet;
and C is a hydrophilic targeting ligand, wherein the hydrophilic targeting
ligand is a LLP2A
prodrug, LLP2A, LXY30, LXW64, DUPA, a LHRH peptide, a HER2 ligand, an EGFR
ligand,
or a toll-like receptor agonist CpG oligonucleotides and wherein when the
hydrophobic moiety is
bis-pyrene, then C is a LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, a LHRH
peptide, an
EGFR ligand, or a toll-like receptor agonist CpG oligonucleotides.
[0089] In some embodiments, the present invention provides a compound of
formula (I): A-B-
C (I), wherein A is a hydrophobic moiety; B is a peptide, wherein the peptide
forms a beta-sheet;
and C is a hydrophilic targeting ligand, wherein the hydrophilic targeting
ligand is a LLP2A
prodrug, LLP2A, LXY30, LXW64, DUPA, folate, a LHRH peptide, a HER2 ligand, an
EGFR
ligand, or a toll-like receptor agonist CpG oligonucleotides and wherein when
the hydrophobic
moiety is bis-pyrene, then C is a LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA,
folate, a
LHRH peptide, an EGFR ligand, or a toll-like receptor agonist CpG
oligonucleotides.
[0090] Hydrophobic moieties useful in the present invention includes any
suitable hydrophobic
moiety known by one of skill in the art. Hydrophobicity and hydrophilicity are
commonly
measured by the log P values of the compounds using the octane-water reference
system. Values
lower than 0 indicate hydrophilicity whereas values higher than 0 indicate
hydrophobicity.
Hydrophobic moieties useful in the present invention includes moieties with
logP values of at
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least 1. In some embodiments, hydrophobic moieties useful in the present
invention have a logP
value of at least 1.5. In some embodiments, hydrophobic moieties useful in the
present invention
have a logP value of 1.5-15. Hydrophobic moieties include, but are not limited
to cholesterol,
vitamin D, vitamin D derivatives, vitamin E, vitamin E derivatives, dyes,
drugs, and radiometal
.. chealators. In some embodiments, the hydrophobic moiety is cholesterol,
vitamin D, vitamin D
derivatives, vitamin E, vitamin E derivatives, a dye, or a drug. In some
embodiments, the
hydrophobic moiety is cholesterol, vitamin D, vitamin E, a dye, or a drug. In
some embodiments,
the hydrophobic moiety is cholesterol, vitamin D, or vitamin E. In some
embodiments, the
hydrophobic moiety is a dye or drug.
.. [0091] Dyes useful in the present invention include any dye described in,
but not limited to,
Johnson, I., Histochemical Journal, 20:123-140 (1998), and The Molecular
Probes Handbook,
11th Edition, ed. Johnson and Spence, Life Technologies, Carlsbad, CA, 2010.
The dyes can be
fluorescent dyes, triarylmethane dyes, cyanine dyes, benzylidene imidazolinone
dyes, indigo
dyes, bis-pyrenes and porphyrins. In some embodiments, the hydrophobic moiety
is a dye. In
some embodiments, the hydrophobic moiety is a fluorescent dye, porphyrin, or
bis-pyrene. In
some embodiments, the hydrophobic moiety is a cyanine dye, porphyrin, or bis-
pyrene.
[0092] Drugs useful in the present invention include chemotherapeutic agents
and
immunomodulcatory agents. For example, the drugs can be, but are not limited
to, deoxycholic
acid, or the salt form deoxycholate, pembrolizumab, nivolumab, cemiplimab, a
taxane (e.g.,
.. paclitaxel, docetaxel, cabazitaxel, Baccatin III, 10-deacetylbaccatin,
Hongdoushan A,
Hongdoushan B, or Hongdoushan C), doxorubicin, etoposide, irinotecan, SN-38,
cyclosporin A,
podophyllotoxin, Carmustine, Amphotericin, Ixabepilone, Patupilone (epothelone
class),
rapamycin and platinum drugs. Other drugs include non-steroidal anti-
inflammatory drugs, and
vinca alkaloids such as vinblastine and vincristine. In some embodiments, the
drug is paclitaxel,
resiquimod, gardiquimod, or deoxycholate.
[0093] In some embodiments, the hydrophobic moiety is a chemotherapeutic
agent, a
fluorescent dye, an immunomodulatory agent, a toll-like receptor agonist, a
small molecule
agonist of stimulator of interferon gene (STING), porphyrin, deoxycholate,
cholesterol, vitamin
D, or vitamin E. In some embodiments, the hydrophobic moiety is a
chemotherapeutic agent, a
.. fluorescent dye, an immunomodulatory agent, a small molecule agonist of
stimulator of
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interferon gene (STING), porphyrin, cholesterol, vitamin D, or vitamin E. In
some embodiments,
the hydrophobic moiety is a chemotherapeutic agent, a fluorescent dye, an
immunomodulatory
agent, a small molecule agonist of stimulator of interferon gene (STING),
porphyrin or
deoxycholate. In some embodiments, the hydrophobic moiety is a
chemotherapeutic agent, a
fluorescent dye, an immunomodulatory agent, porphyrin or deoxycholate. In some
embodiments,
the hydrophobic moiety is paclitaxel, bis-pyrene, cyanine dye, resiquimod,
gardiquimod,
amidobenzimidazole, porphyrin, or deoxycholate. In some embodiments, the
hydrophobic
moiety is paclitaxel, bis-pyrene, cyanine dye, resiquimod, gardiquimod,
porphyrin, or
deoxycholate. In some embodiments, the hydrophobic moiety is resiquimod or
porphyrin.
[0094] Porphyrins useful in the present invention include any porphyrin known
by one of skill
in the art. In some embodiments, the porphyrin is a substituted or
unsubstituted porphin,
protoporphyrin IX, octaethylporphyrin, tetraphenyl porphyrin, pyropheophorbide-
a,
pheophorbide, chlorin e6, purpurin or purpurinimide. In some embodiments, the
porphyrin is
pyropheophorbide-a, pheophorbide, chlorin e6, purpurin or purpurinimide. In
some
embodiments, the porphyrin is pheophorbide-a. In some embodiments, the
porphyrin has the
following structure:
Z
0
`N. 0
'
[0095] In some embodiments, the hydrophobic moiety is bis-pyrene. Bis-pyrenes
useful in the
present invention include any bis-pyrene known by one of skill in the art. In
some embodiments,
the bis-pyrene comprises the following moieties:
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I
/ I
1 I 1 1 I 1
w -...w,,, / W
I ¨
1 0 1 0 1 ,õ.,=======.."'%"\,../\,,,
I N
I / N \ I fi /
/ .1 /
.
I
\ / and W
.
In some embodiments, the bis-pyrene comprises the following:
lie 1). Alk.
0 0
4I 1 41 1 N / \ 1
0 0
4/411 40.11 44
. . .
, Or
, .
In some embodiments, the bis-pyrene has the following structure:
0
0
0
40.11
=
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[0096] The peptides useful in the present invention can be any suitable
peptide, and have any
suitable peptide sequence length known by one of skill in the art. In some
embodiments, the
peptide is a peptide sequence 5-50 amino acids in length. In some embodiments,
the peptide is a
peptide sequence 5-40 amino acids in length. In some embodiments, the peptide
is a peptide
sequence 5-30 amino acids in length. In some embodiments, the peptide is a
peptide sequence 5-
25 amino acids in length. In some embodiments, the peptide is a peptide
sequence 5-20 amino
acids in length. In some embodiments, the peptide is a peptide sequence 5-15
amino acids in
length. In some embodiments, the peptide is a peptide sequence about 5-10
amino acids in
length.
[0097] Adjacent beta-strand peptides form hydrogen bonds between each strand
resulting in
beta sheet peptides. The beta-sheet peptide sequences useful in the present
invention can be any
suitable peptide sequence known by one of skill in the art. For example,
commonly known beta-
sheet peptides are described in "Branched KLVFF tetramers strongly potentiate
inhibition of
beta-amyloid aggregation" (Chafekar et al., Chembiochem. 2007 Oct
15;8(15):1857-64). In some
embodiments, the peptide comprises a peptide sequence from a beta-sheet
peptide domain of
green fluorescent protein, interleukins, immunoglobulins, or beta-amyloid
peptide. In some
embodiments, the peptide comprises a peptide sequence from a beta-sheet
peptide domain of a
beta-amyloid peptide. In some embodiments, the beta-amyloid peptide is beta-
amyloid 40 or
beta-amyloid 42. In some embodiments, the beta-amyloid peptide is beta-amyloid
40.
[0098] In some embodiments, the peptide comprises at least 40% sequence
identity to SEQ ID
NO: 1. In some embodiments, the peptide comprises at least 50% sequence
identity to SEQ ID
NO: 1. In some embodiments, the peptide comprises at least 60% sequence
identity to SEQ ID
NO: 1. In some embodiments, the peptide comprises at least 80% sequence
identity to SEQ ID
NO: 1. In some embodiments, the peptide comprises SEQ ID NO: 1.
[0099] In some embodiments, the peptide comprises at least 40% sequence
identity to SEQ ID
NO:2. In some embodiments, the peptide comprises at least 50% sequence
identity to SEQ ID
NO:2. In some embodiments, the peptide comprises at least 60% sequence
identity to SEQ ID
NO:2. In some embodiments, the peptide comprises at least 80% sequence
identity to SEQ ID
NO:2. In some embodiments, the peptide comprises SEQ ID NO:2.
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[0100] In some embodiments, the peptide comprises at least 40% sequence
identity to SEQ ID
NO:3. In some embodiments, the peptide comprises at least 50% sequence
identity to SEQ ID
NO:3. In some embodiments, the peptide comprises at least 60% sequence
identity to SEQ ID
NO:3. In some embodiments, the peptide comprises at least 80% sequence
identity to SEQ ID
NO:3. In some embodiments, the peptide comprises SEQ ID NO:3.
[0101] Hydrophilic targeting ligands useful in the present invention can
target receptors on the
cell surface, or the acellular component of the tumor microenvironment.
Hydrophilicity and
hydrophobicity are commonly measured by the log P values of the compounds
using the octane-
water reference system. Values lower than 0 indicate hydrophilicity whereas
values higher than 0
indicate hydrophobicity. In some embodiments, the hydrophilic targeting ligand
includes
peptides which target cell surface receptors or acellular components in the
tumor
microenvironment, which includes, but is not limited to immune cells such as
macrophages, T
cells, and B cells. In some embodiments, the hydrophilic targeting ligand
targets cell surface
receptors such as, but not limited to, integrins and epidermal growth factor
receptors. In some
embodiments, the hydrophilic targeting ligand targets integrins, epidermal
growth factors, and
toll-like receptors.
[0102] In some embodiments, the hydrophilic targeting ligand is a HER2 ligand,
a prodrug for
a HER2 ligand, a receptor tyrosine-protein kinase-targeting ligand, an
integrin-targeting ligand,
epidermal growth factor receptor-targeting ligand, ovarian cancer cell-
targeting ligand, or
prostate cancer cell-targeting ligand. In some embodiments, the hydrophilic
targeting ligand is a
HER2 ligand, a prodrug for a HER2 ligand, an integrin-targeting ligand,
epidermal growth factor
receptor-targeting ligand, ovarian cancer cell targeting ligand, or prostate
cancer cell targeting
ligand.
[0103] In some embodiments, the hydrophilic targeting ligand is a HER2 ligand.
In some
embodiments, the HER2 ligand is an anti-HER2 antibody peptide. In some
embodiments, the
hydrophilic targeting ligand is the HER2 ligand, wherein the HER2 ligand is an
anti-HER2
antibody peptide mimic derived from the primary sequence of the CDR-H3 loop of
the anti-
HER2 rhumAb 4D5. In some embodiments, the HER2 ligand is as described in
"Rationally
designed anti-HER2/neu peptide mimetic disables P185HER2/neu tyrosine kinases
in vitro and
in vivo" (Park et al. Nat Biotechnol. 2000 Feb;18(2):194-8.)
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[0104] In some embodiments, the HER2 ligand has at least 40% sequence identity
to SEQ ID
NO:4. In some embodiments, the HER2 ligand has at least 50% sequence identity
to SEQ ID
NO:4. In some embodiments, the HER2 ligand has at least 60% sequence identity
to SEQ ID
NO:4. In some embodiments, the HER2 ligand has at least 80% sequence identity
to SEQ ID
NO:4. In some embodiments, the HER2 ligand is SEQ ID NO:4.
[0105] In some embodiments, the hydrophilic targeting ligand is an integrin-
targeting ligand,
epidermal growth factor receptor-targeting ligand, ovarian cancer cell
targeting ligand, or
prostate cancer cell targeting ligand. In some embodiments, the hydrophilic
targeting ligand is a
prodrug for an integrin-targeting ligand, epidermal growth factor receptor-
targeting ligand,
ovarian cancer cell targeting ligand, or prostate cancer cell targeting
ligand.
[0106] In some embodiments, the hydrophilic targeting ligand is a LLP2A
prodrug, LLP2A,
LXY30, DUPA, folate, a LHRH peptide, or an EGFR ligand. Any one of the
carboxylic acid
groups in the DUPA structure can be used to link to the beta-sheet peptide.
LHRH analog
peptide comprises the following peptide sequence: H-Glp-His-Trp-S er-Thr-Lys-
Leu-Arg-Pro-
Gly-NH2 or H-Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH2. The Lys side chain
NH2 group
of the LHRH peptides can be used to link to the beta-peptide sheet. In some
embodiments, the
NH2 group is used to covalently link to the beta-peptide sheet.
[0107] EGFR ligands useful in the present invention includes any EGFR ligand
known by one
of skill in the art. In some embodiments, the EGFR ligand can be EGF, TGF-
alpha, HB-EGF,
amhiregulin, betacellulin, epigen, epiregulin, neuregulin 1, neuregulin 2,
neuregulin 3, and
neuregulin 4.
[0108] In some embodiments, the hydrophilic targeting ligand is a LLP2A
prodrug, LLP2A, or
LXY30. The LLP2A prodrug can include any cleavable functional group to be
metabolized in
situ known by one of skill in the art. In some embodiments, the LLP2A prodrug
comprises an
ester, amide, carbamate, oxime, imine, ether, phosphate, or beta-amino-ketone
functional group.
In some embodiments, the LLP2A prodrug comprises an ester, amide, carbamate,
ether, or
phosphate functional group. In some embodiments, the LLP2A prodrug comprises
an ester,
amide, carbamate or phosphate functional group. In some embodiments, the LLP2A
prodrug
comprises an ester group.
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[0109] In some embodiments, the hydrophilic targeting ligand is a LLP2A
prodrug, with the
following structure:
pc H3
0. "
1 di
0 0
N jt, N LA, lies
i N
0 ..... , 0
N,... I NH
0
In some embodiments, the hydrophilic targeting ligand is LLP2A, with the
following structure:
COOH
it,A0 ,11c3tOss:,
40 riIN 0 PI 0 1 14 0
N-... / NH
.---
5 0
In some embodiments, the hydrophilic targeting ligand is LXY30, with the
following structure:
liNyN112
_NH
i
,CONH20 (..)
H2N 0 0 0
A3.4
I 1 WI 11 '%ici 11 11; 3
-cooH
i--)NA,µ lici
/
F
S S/
[0110] In some embodiments, the compound of the present invention has the
following
structure:
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/
---. -... \
\ NH N-
HNyNH2
\ i
NH N HN
t
*/
CONH20 0
H2N 0 H 0 0 0
ItykryNylINN-"'yiNyli-trIKI N ___________________ ICKL3iFFK 0
-COOH
* HO H2N---*--:0
F F
S S
[0111] In some embodiments, the compound of the present invention has the
following
structure:
PCK-i
q õ...._./
'..= '
1\ d
,L.õ()..-:;r.
f
.
N:
Ili N KLVFFK ________________________________________ \
ti;'s==::
iis õ.011, i N N
,I. 0 ; 0
N N H2N ---0
'-....1)
----
0
[0112] In some embodiments, the compound of the present invention has the
following
structure:
C00 0 \ ,..,0-1-
il
1-1
N = .
s s:
N.: ..'. .
1111 I
N N H2N -0
,--
1 ...)
N ,. NH
0
[0113] In some embodiments, the compound of the present invention has the
following
structure:
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COOH
\ /
H 0 0
0
* 1 H 0
1 N
0
N N HgeL-0 \
NraH
..,-.'
0
[0114] In some embodiments, the compound of the present invention has the
following
structure:
HNNH2
1
"
cONH20 4 0 =,.
l' = '
"
.,
H2N 14 0 i
1.4 0
N----r- , N------K-P-N4k1 H KLVFFK e s. .6
M1::
,
H 0 i H 8y H 0 1 0 .
COOH
. HO H2N 0
S F F S
[0115] In some embodiments, the compound of the present invention has the
following
structure:
1111,
1)11
0
. 0 F FVL KYC DG
FYACYM DV
0
0
40.11
ilk .
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W. NANOCARRIERS
[0116] In some embodiments, the present invention provides a nanocarrier
having an interior
and an exterior, the nanocarrier comprising a plurality of compounds of the
present invention,
wherein each compound self-assembles in an aqueous solvent to form the
nanocarrier such that a
hydrophobic pocket is formed in the interior of the nanocarrier, and a
hydrophilic group self-
assembles on the exterior of the nanocarrier.
[0117] The diameter of the nanocarrier of the present invention can be any
suitable size known
by one of skill in the art. In some embodiments, the nanocarrier can have a
diameter of 5 to 100
nm. In some embodiments, the nanocarrier can have a diameter of 10 to 100 nm.
In some
embodiments, the nanocarrier can have a diameter of 15 to 80 nm. In some
embodiments, the
nanocarrier can have a diameter of 25 to 60 nm. In some embodiments, the
nanocarrier can have
a diameter of about 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, or about 70 nm. In some
embodiments,
the nanocarrier can have a diameter of about 20 nm or about 30 nm. In some
embodiments, the
nanocarrier can have a diameter of about 20 nm. In some embodiments, the
nanocarrier can have
a diameter of about 30 nm.
[0118] The exterior of the nanocarrier can be used for cell targeting. The
nanocarrier of the
present invention can target cell surface receptors and proteins such as, but
not limited to
integrins, human epidermal growth factor receptor 2 (HER2), epidermal growth
factor receptors,
and G protein-coupled receptors. In some embodiments, the nanocarrier can
target integrins and
HER2.
[0119] The nanocarrier can transform in situ after binding to the receptors or
proteins on the
cell surface to form a nanofibrillar structure. In some embodiments, the
nanocarrier can
transform in situ after binding to HER2 on the cell surface.
[0120] In some embodiments, the nanocarrier further comprises a hydrophobic
drug or an
imaging agent sequestered in the hydrophobic pocket of the nanocarrier.
[0121] The hydrophobic drugs useful in the present invention can be any
hydrophobic drug
known by one of skill in the art. Hydrophobic drugs useful in the present
invention include, but
are not limited to, deoxycholic acid, deoxycholate, resiquimod, gardiquimod,
imiquimod, a
taxane (e.g., paclitaxel, docetaxel, cabazitaxel, Baccatin III, 10-
deacetylbaccatin, Hongdoushan
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A, Hongdoushan B, or Hongdoushan C), doxorubicin, etoposide, irinotecan, SN-
38, cyclosporin
A, podophyllotoxin, Carmustine, Amphotericin, Ixabepilone, Patupilone
(epothelone class),
rapamycin and platinum drugs. Other drugs includes non-steroidal anti-
inflammatory drugs, and
vinca alkaloids such as vinblastine and vincristine.
[0122] The imaging agents useful in the present invention can be any imaging
agent known by
one of skill in the art. Imaging agents include, but are not limited to,
paramagnetic agents, optical
probes, and radionuclides. Paramagnetic agents are imaging agents that are
magnetic under an
externally applied field. Examples of paramagnetic agents include, but are not
limited to, iron
particles including nanoparticles. Optical probes are fluorescent compounds
that can be detected
by excitation at one wavelength of radiation and detection at a second,
different, wavelength of
radiation. Optical probes useful in the present invention include, but are not
limited to, Cy5.5,
Alexa 680, Cy5, DiD (1,1'-dioctadecy1-3,3,3',3'-tetramethylindodicarbocyanine
perchlorate) and
DiR (1,1'-dioctadecy1-3,3,3',3'-tetramethylindotricarbocyanine iodide). Other
optical probes
include quantum dots. Radionuclides are elements that undergo radioactive
decay.
Radionuclides useful in the present invention include, but are not limited to,
3H, HC, 13N, 18F,
19F, 60co, 6401, 67¨u,
68Ga, 82Rb, 90Sr, 90Y, 99Tc, 99mTc, '''In, 1231, 1241, 125j, 1291, 1311,
137cs, 177Lti,
186,,K e,
188Re, 211At, Rn, Ra, Th, U, Pu and 241Am.
[0123] The nanocarrier can include a plurality of conjugates. For example, the
nanocarrier can
include a plurality of two, three, four, five, six, or more, different
conjugates. In some
embodiments, the nanocarrier comprises a plurality of two different
conjugates. In some
embodiments, the nanocarrier comprises a plurality of three different
conjugates. In some
embodiments, the nanocarrier comprises a plurality of four different
conjugates.
[0124] In some embodiments, the present invention provides a nanocarrier
having an interior
and an exterior, the nanocarrier comprising a plurality of a first conjugate
and a second conjugate
wherein the first conjugate comprises formula (I): A-B-C (I); and the second
conjugate
comprises formula (II): A'-B'-C' (II) wherein: A and A' are each independently
a hydrophobic
moiety; B and B' are each independently a peptide, wherein each peptide
independently forms a
beta-sheet; and C and C' are each independently a hydrophilic targeting
ligands, wherein each
hydrophilic targeting ligand is independently a LLP2A prodrug, LLP2A, LXY30,
LXW64,
DUPA, folate, a LHRH peptide, a HER2 ligand, an EGFR ligand, or a radiometal
chelator; and
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wherein A and A' are different hydrophobic moieties and/or C and C' are
different hydrophilic
targeting ligands.
[0125] In some embodiments, the nanocarrier comprises a plurality of a first
conjugate and a
second conjugate as described above, and further comprises a third conjugate
comprising
formula (III): A"-B"-C" (III) wherein A" is a hydrophobic moiety, B" is a
peptide, wherein the
peptide forms a beta __ sheet, and C" is a hydrophilic targeting ligand, and
wherein A, A', and A"
are different hydrophobic moieties and/or C, C', and C" are different
hydrophilic targeting
ligands. In some embodiments, the nanocarrier further comprises a fourth, a
fifth, or a sixth
conjugate where each additional conjugate is independently of formula III.
[0126] The nanocarrier of the present invention can comprise a plurality of
two different
conjugates. The nanocarriers comprising a plurality of two different
conjugates can have
diameters as described above. The nanocarriers comprising a plurality of two
different
conjugates can have similar targeting and transformative properties as
described above.
[0127] Suitable hydrophobic moieties for the nanocarriers of the present
invention are
described above. In some embodiments, each hydrophobic moiety is independently
a dye, a drug,
or a radiometal chelator. In some embodiments, each hydrophobic moiety is
independently a bis-
pyrene, porphyrin, resiquimod, or gardiquimod.
[0128] In some embodiments, each hydrophobic moiety is independently a
porphyrin or
resiquimod. In some embodiments, the porphyrin is pyropheophorbide-a,
pheophorbide, chlorin
e6, purpurin or purpurinimide. In some embodiments, the porphyrin is
pheophorbide-a. In some
embodiments, the porphyrin has the following structure:
Z
0
\ 0 .
[0129] In some embodiments, the resiquimod has the following structure:
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NH2
N
N "=-=
N
\--(01.1
[0130] Radiometal chelators useful in the present invention include any
radiometal chelator
known by one of skill in the art. In some embodiments, the radiometal chelator
is a Gd(III)
chelator, diethylenetriaminepentaaetic anhydride (DTPA), 1,4,8,11-
tetraazacyclotetradecane-
1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-
tetracetic acid
(DOTA), or 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). In some
embodiments, the
radiometal chelator is a Gd(III) chelator, DOTA chelator, or a NOTA chelator.
[0131] Suitable peptide sequence lengths for the nanocarriers of the present
invention are
described above. In some embodiments, each peptide is independently a peptide
sequence 5-30
amino acids in length. In some embodiments, each peptide is independently a
peptide sequence
5-25 amino acids in length. In some embodiments, each peptide is independently
a peptide
sequence 5-20 amino acids in length.
[0132] Suitable peptide sequence for the nanocarriers of the present invention
are described
above. In some embodiments, each peptide independently comprises a peptide
sequence from a
beta-sheet peptide domain of a beta-amyloid peptide. In some embodiments, the
beta-amyloid
peptide is beta-amyloid 40 or beta-amyloid 42. In some embodiments, the beta-
amyloid peptide
is beta-amyloid 40.
[0133] In some embodiments, each peptide independently comprises at least 40%
sequence
identity to SEQ ID NO: 1. In some embodiments, each peptide independently
comprises at least
50% sequence identity to SEQ ID NO: 1. In some embodiments, each peptide
independently
comprises at least 60% sequence identity to SEQ ID NO: 1. In some embodiments,
each peptide
independently comprises at least 80% sequence identity to SEQ ID NO: 1. In
some embodiments,
each peptide independently comprises SEQ ID NO: 1.
[0134] In some embodiments, each peptide independently comprises at least 40%
sequence
identity to SEQ ID NO:2. In some embodiments, each peptide independently
comprises at least
50% sequence identity to SEQ ID NO:2. In some embodiments, each peptide
independently
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comprises at least 60% sequence identity to SEQ ID NO:2. In some embodiments,
each peptide
independently comprises at least 80% sequence identity to SEQ ID NO:2. In some
embodiments,
[0135] Suitable hydrophilic targeting ligands for the nanocarriers of the
present invention are
described above. In some embodiments, each hydrophilic targeting ligand is
independently a
LLP2A prodrug, LLP2A, LXY30, folate, a LHRH peptide, a HER2 ligand, an EGFR
ligand, a
Gd(III) chelator, a DOTA chelator, or a NOTA chelator. In some embodiments,
each hydrophilic
targeting ligand is independently a LLP2A prodrug, LLP2A, LXY30, a LHRH
peptide, a HER2
ligand, an EGFR ligand, a DOTA chelator, or a NOTA chelator. In some
embodiments, each
hydrophilic targeting ligand is independently a LLP2A prodrug, LLP2A or LXY30.
[0136] In some embodiments, each hydrophilic targeting ligand is independently
a LLP2A
prodrug, with the following structure:
pcm,
0
ILik,
=N; so
0
0
N
N NH
[0137] In some embodiments, each hydrophilic targeting ligand is independently
LLP2A, with
the following structure:
COOH
0 I;L)t_ 4/460ss.
N
N 0
/ NH
[0138] In some embodiments, each hydrophilic targeting ligand is independently
LXY30, with
the following structure:
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HN.1...õN112
.õNH
1
õCON1120 (
H2N L, 0 L, 0 Q ( H
II tnif i p Fi(jpb
\\() 'COOH /
\S , =--, F ... S/ .
[0139] In some embodiments, the first conjugate has the structure:
."
CONH20
0 0
--, ---,. \
NH N
\
N.,c
H2N;NH2
FI ¨
1 \ i
,eH N HN
i
N 1 ,(1,r1Lkly t
10/
14
0 N101- 1 (1p N 0
'COOH
so HO / IFICKLVF
FI2N-- 0
F F
S S .
[0140] In some embodiments, the second conjugate has the structure:
pc H3
q , .. ?
./
0 , ,
o\..,0--,';''' ---=,,,.
õ::::::.'":,
==:=:.'
, . .
. ..
14 0 14 0 ,
:, ::\;.=
",
'
N;.i.:=':.2
IP NIN 0 ______ ,.,, \ 4",:. t:: te:::
"Nejl... ks.:,...V: :: ,,,
i N 0 ,(õ \\0
H2N ----.0
....- )----1
N-ra..,...õ--14 NH
.....-- r-
.
[0141] In some embodiments, the second conjugate is converted in situ to the
following
structure:
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COOH0.
0
6KLVEFK
0 0
11 N
H2N
N NH
0
[0142] The ratio of the first conjugate to the second conjugate of the
nanocarriers of the
present invention can be any suitable ratio known by one of skill in the art.
In some
embodiments, the ratio of the first conjugate to the second conjugate is about
25:1 to 1:25. In
some embodiments, the ratio of the first conjugate to the second conjugate is
about 25:1 to 1:10.
In some embodiments, the ratio of the first conjugate to the second conjugate
is about 10:1 to
about 1:10. In some embodiments, the ratio of the first conjugate to the
second conjugate is about
10:1, 8:1, 5:1, 3:1, or 1:1. In some embodiments, the ratio of the first
conjugate to the second
conjugate is about 1:1.
V. NANOFIBRILS
[0143] In some embodiments, the present invention provides a method of forming
nanofibrils,
comprising contacting a nanocarrier of the present invention with a cell
surface or acellular
component at a tumor microenvironment, wherein the nanocarrier undergoes in
situ
transformation to form fibrillary structures, thereby forming the nanofibrils.
[0144] When the nanocarrier of the present invention binds with the cell
surface or acellular
component at a tumor microenvironment, it can undergo an in situ
transformation to form
nanofibrils, which can disrupt the cells and/or the tumor microenvironment.
Transformation of
the nanocarrier occurs when the hydrophilic targeting ligands of the
nanocarriers bind to the cell
surface or acellular component of interest, triggering formation of fibrillary
structures which
form the nanofibrils.
[0145] The tumor microenvironment comprises tumor cells and the surrounding
environment,
including, but is not limited to, the extracellular matrix, infiltrating host
cells, secreted factors,
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signaling molecules, immune cells, stromal cells, dendritic cells, T cells,
myeloid derived
suppressor cells, vasculature, blood cells, cytokines, chemokines, growth
factors, fibroblast and
macrophages, any of which the nanocarrier of the present invention can
interact with to form
nanofibrils.
[0146] Nanocarriers of the present invention can form highly ordered beta-
sheet fibrillary
structures of the nanofibrils. Without being bound by any particular theory,
one possible
explanation for forming the beta-sheet fibrillary structures is that the beta-
sheet forming peptides
in the conjugates influence formation of the beta-sheet fibrillary structures
of the nanofibrils.
[0147] Nanofibrils of the present invention can have any suitable diameter
known by one of
.. skill in the art. In some embodiments, the diameter of the nanofibril is 5
to 50 nm. In some
embodiments, the diameter of the nanofibril of the nanofibril is 5 to 30 nm.
In some
embodiments, the diameter of the nanofibril is 5 to 15 nm. In some
embodiments, the diameter of
the nanofibril is 5 to 10 nm. In some embodiments, the diameter of the
nanofibril is about 5 nm,
6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, or about 12 nm.
[0148] Transformation of the nanocarrier to the nanofibril can be determined
by imaging
techniques known by one of skill in the art and by measuring the particle size
of the nanocarrier.
For example, transformation of the nanocarrier to nanofibril can be determined
using TEM
imaging wherein the round nanocarrier shapes are transformed into nanofibril
structures
following binding of the nanocarrier to the cell surface or acellular
component at a tumor
microenvironment. In another example, nanocarrier size can be determined using
dynamic light
scattering (DLS). In DLS studies, when the nanocarrier is transformed into
nanofibrils, the peak
around the diameter of a nanocarrier, for example 10-100 nm, will decrease
over time, as the
peak around 500 nm-1000 nm increase over time, indicating formation of the
nanofibrils.
VI. METHOD OF TREATMENT AND IMAGING
.. [0149] In some embodiments, the present invention provides a method of
treating a disease,
comprising administering to a subject in need thereof, a therapeutically
effective amount of a
nanocarrier of the present invention, wherein the nanocarrier forms
nanofibrils in situ after
binding to a cell surface or acellular component at the tumor
microenvironment, thereby treating
the disease.
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[0150] Binding to the cell surface or acellular component can be determined by
one of
ordinary skill in the art using fluorescence microscopy. Binding to the cell
surface or acellular
component can be determined when the nanocarrier comprises conjugates with a
fluorescent dye
as the hydrophobic moiety and the cell is labeled with any fluorescent dye
known by one of skill
in the art. One of skill in the art can select a suitable dye to use based on
which fluorescent dye is
used as the hydrophobic moiety. For example, when the nanocarrier comprises
conjugates
wherein the hydrophobic moiety comprises bis-pyrenes, which is a green
fluorescent dye, then
the cell can be labeled with a non-green fluorescent dye, such as, but not
limited to, a red
fluorescent dye or a blue fluorescent dye. In another example, if the
hydrophobic moiety
comprises a red fluorescent dye, such as, but not limited to, porphyrin, then
one of skill in the art
can chose a non-red fluorescent dye, such as a green fluorescent dye or blue
fluorescent dye.
[0151] The tumor microenvironment comprises tumor cells and the surrounding
environment,
including, but not limited to, the extracellular matrix, infiltrating host
cells, secreted factors,
signaling molecules, immune cells, stromal cells, dendritic cells, T cells,
myeloid derived
suppressor cells, vasculature, blood cells, cytokines, chemokines, growth
factors, fibroblast and
macrophages. Tumor growth and progression can be influenced by interactions of
the cancer
cells with the microenvironment, which can result in eradication of cancer
cells, metastasize of
cancer cells, or establishing dormant micrometastases cancer cells. The tumor
microenvironment
can be targeted for therapeutic responses.
[0152] Binding to the acellular component at the tumor microenvironment
includes, but is not
limited to, binding to the proteins within the extracellular matrix and other
ligands, compounds,
or dendritic cells which are directly attached to the tumor cell or
surrounding cells.
[0153] The nanocarriers of the present invention can be administered to a
subject for treatment,
of diseases including cancer such as, but not limited to: carcinomas, gliomas,
mesotheliomas,
melanomas, lymphomas, leukemias, adenocarcinomas, breast cancer, ovarian
cancer, cervical
cancer, glioblastoma, leukemia, lymphoma, prostate cancer, and Burkitfs
lymphoma, head and
neck cancer, colon cancer, colorectal cancer, non-small cell lung cancer,
small cell lung cancer,
cancer of the esophagus, stomach cancer, pancreatic cancer, hepatobiliary
cancer, cancer of the
gallbladder, cancer of the small intestine, rectal cancer, kidney cancer,
bladder cancer, prostate
cancer, penile cancer, urethral cancer, testicular cancer, cervical cancer,
vaginal cancer, uterine
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cancer, ovarian cancer, thyroid cancer, parathyroid cancer, adrenal cancer,
pancreatic endocrine
cancer, carcinoid cancer, bone cancer, skin cancer, retinoblastomas, multiple
myelomas,
Hodgkin's lymphoma, and non-Hodgkin's lymphoma (see, CANCER: PRINCIPLES AND
PRACTICE (DeVita, V. T. et al. eds 2008) for additional cancers).
[0154] Other diseases that can be treated by the nanocarriers of the present
invention include:
(1) inflammatory or allergic diseases such as systemic anaphylaxis or
hypersensitivity responses,
drug allergies, insect sting allergies; inflammatory bowel diseases, such as
Crohn's disease,
ulcerative colitis, ileitis and enteritis; vaginitis; psoriasis and
inflammatory dermatoses such as
dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria;
vasculitis;
spondyloarthropathies; scleroderma; respiratory allergic diseases such as
asthma, allergic
rhinitis, hypersensitivity lung diseases, and the like, (2) autoimmune
diseases, such as arthritis
(rheumatoid and psoriatic), osteoarthritis, multiple sclerosis, systemic lupus
erythematosus,
diabetes mellitus, glomerulonephritis, and the like, (3) graft rejection
(including allograft
rejection and graft-v-host disease), and (4) other diseases in which undesired
inflammatory
responses are to be inhibited (e.g., atherosclerosis, myositis, neurological
conditions such as
stroke and closed-head injuries, neurodegenerative diseases, Alzheimer's
disease, encephalitis,
meningitis, osteoporosis, gout, hepatitis, nephritis, sepsis, sarcoidosis,
conjunctivitis, otitis,
chronic obstructive pulmonary disease, sinusitis and Behcet's syndrome).
[0155] In some embodiments, the disease is cancer. In some embodiments, the
disease is
selected from the group consisting of bladder cancer, brain cancer, breast
cancer, cervical cancer,
cholangiocarcinoma, colorectal cancer, esophageal cancer, gall bladder cancer,
gastric cancer,
glioblastoma, intestinal cancer, head and neck cancer, leukemia, liver cancer,
lung cancer,
melanoma, myeloma, ovarian cancer, pancreatic cancer and uterine cancer. In
some
embodiments, the disease is selected from the group consisting of bladder
cancer, breast cancer,
colorectal cancer, esophageal cancer, glioblastoma, head and neck cancer,
leukemia, lung cancer,
myeloma, ovarian cancer, and pancreatic cancer.
[0156] In some embodiments, the nanocarrier of the present invention can be
used for
combination therapy. In some embodiments, the combination therapy includes a
nanocarrier of
the present invention and at least one checkpoint inhibitor. Representative
checkpoint inhibitors
include, but are not limited to, anti-CTLA-4 therapy, an anti-PD-1 therapy, or
an anti-PD-Li
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therapy, for example. Examples include ipilimumab, nivolumab, pembrolizumab,
pidilizumab,
atezolizumab, Ipilimumab, and/or tremelimumab, and may include combination
therapies, such
as nivolumab+ipilimumab.
[0157] In some embodiments, the present invention provides a method of
imaging, comprising
administering to a subject to be imaged, an effective amount of a nanocarrier
of the present
invention.
[0158] Suitable imaging agents for the nanocarriers of the present invention
are described
above. For example imaging agents include, but are not limited to,
paramagnetic agents, optical
probes, and radionuclides. Opitcal probes include, but are not limited to
fluorescent dyes such as
cyanine dyes, bis-pyrenes, and porphyrin.
VII. EXAMPLES
Example 1: Nanocarriers of BP-FFVLK-YCDGFYACYMDV
[0159] This example describes design and synthesis of a smart supramolecular
peptide, BP-
FFVLK-YCDGFYACYMDV, capable of (1) assembling into nanoparticles (NPs) under
aqueous
condition and in blood circulation, and (2) in situ transformation into
nanofibrillar (NFs)
structure upon binding to the cell surface HER2 at the tumour sites. This
transformable peptide
monomer (TPM), a supramolecular material, was comprised of three discrete
functional
domains: (1) the bis-pyrene (BP) moiety with aggregation induced emission
(ATE) property for
fluorescence reporting, and as a hydrophobic core to induce the formation of
micellar NPs, (2)
the KLVFF 3-sheet forming peptide domain originated from P-amyloid (AP)
peptide, and (3) the
YCDGFYACYMDV disulfide cyclic peptide HER2-binding domain, an anti-HER2/neu
antibody peptidic mimic derived from the primary sequence of the CDR-H3 loop
of the anti-
HER2 rhumAb 4D5. Under aqueous condition, the supramolecular peptide would
self-assemble
into spherical NPs, in which BP and KLVFF domains constituted the hydrophobic
core and
YCDGFYACYMDV peptide constituted the negatively charged hydrophilic corona.
NPs,
injected intravenously (i.v.) into mice bearing HER2+ tumours, were found to
be preferentially
accumulated at the tumour site. Upon interaction with HER2 displayed on the
tumour cell
surface, the NPs would undergo in situ transformation into fibrillar
structural network, with long
retention time. Such HER2 binding extracellular fibrillar network was found to
greatly suppress
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the dimerization of HER2 and prevent downstream cell signaling and expression
of proliferation
and survival genes in the nucleus. These structural transformation-based
supramolecular peptides
represent a novel class of receptor-mediated targeted therapeutics against
cancers.
Materials and Methods
[0160] The preparation of transformable peptide monomers (TPMS) 1'-4'. The
hydrophobic bis-pyrene unit (BP-COOH) was synthetized as previously reported
(Qiao, S.-L. et
al. Thermo-Controlled in Situ Phase Transition of Polymer¨Peptides on Cell
Surfaces for High-
Performance Proliferative Inhibition. ACS Appl. Mater. Interfaces 8, 17016-
17022 (2016).). The
TPMs l'-4' were synthesized by standard solid phase peptide synthesis
techniques. The BP-
COOH as a hydrophobic part was linked to TPMs 1'-4' chain. For TPMs 3' and 4',
PEG1000 as
a hydrophilic unit was linked to the peptide to replace HER2 ligand of
molecules 1 and 2. The
molecular structures of BP dye and peptides were confirmed by matrix-assisted
laser desorption
ionization time-off light mass spectrometry (ESI and MALDI-TOF mass spectra,
Bruker
Daltonics).
[0161] Self-assembly preparation and characterization of NPs. The TPMs 1'-4'
were
dissolved in DMSO to form a solution, respectively. Peptide solution (5 pL)
was further diluted
with DMSO (995, 795, 595, 395, 195, 95, 15, 0 L) and mixed with deionized
water (0, 200, 400,
600, 800, 900, 980, 995 pL), respectively. The UV¨vis absorption and
fluorescence spectra
(Thermo Scientific, Waltham, MA) of different water content mixture solution
were measured to
validate the formation of NPs. The fresh NPs (99% water content, 20 laM) were
used for the
measurement as an initial state. The morphology transformation of NPs to NFs
was administrated
by adding HER2 extracellular receptor protein (expressed in HEK 293 cells,
Sigma-Aldrich) and
cultured for several hours at 37 C. At different time point (0.5, 6 and 24
h), the solution was
used for size/zeta potential (Microtrac, America), CD (JASCO Inc, Easton, MD,
USA), and TEM
measurement (Philips CM-120 TEAL America). The TEM sample was dyed by uranyl
acetate.
[0162] Stability of NPs1 in human plasma. The stability of NPs1 was studied in
10 % (v/v)
plasma from healthy human volunteers. The mixture was incubated at
physiological body
temperature (37 C) followed by size measurements at predetermined time
intervals up to 168 h.
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[0163] MCF-7/C6 cells induction process. The induction method of MCF-7/C6
cells was
obtained from Professor Jim Jim Li's lab (Departments of Radiation Oncology,
University of
California Davis), The MCF-7/C6 radioresistant cell line was survived from 25
fractionated
ionizing radiations with a total dose of 50 Gy y rays (2 Gy per fraction, five
times per week).
[0164] CLSM and SEM validation of NPs structural transformation on cell
surfaces. The
cells were cultured in glass bottom dishes for 12 h. NPs1-4 (50 pM) was
incubated with cells in
DMEM at 37 C for 0.5, 6 and 24 h, respectively. For confocal laser scanning
microscope
(CLSM, Zeiss LSM710, Jena, Germany) imaging, the specimens were solidified
with
glutaraldehyde (4%) for 10 min, washed with PBS for 3 times and examined with
a 40x or 63x
immersion objective lens using a 405 nm laser. To further validate the binding
of NPs1 to HER2,
rabbit anti-HER2 (29D8) monoclonal antibody (MAb) (Sigma Aldrich, USA) was
used to detect
the extracellular domain of HER2 on the surface of MCF-7/C6 cells. For SEM
(Philips XL30
TMP, FEI Company, Hillsboro), the cells were solidified with glutaraldehyde
(4%) overnight
and then coated with gold for 2 mM.
[0165] In vitro cytotoxic assay. MCF-7/C6, MCF-7, SKBR-3 and BT474 cells were
used to
evaluate the cytotoxicity of NPs1-4. Cells per well were seeded in the 96-well
plates (n = 3)
cultured with DMEM supplemented with 10% FBS and 1% penicillin at 37 C in a
humidified
environment containing 5% CO2. DMS0 solution of 1-4 were diluted by DMEM (1.5,
7.5, 15,
75, 150, 300 laM) and then added into each well to incubate with cells. After
48 h of incubation,
MTS reagent was added into each well. The relative cell viabilities were
measured by Micro-
plate reader (SpectraMax M2). Percentage of cell viability represented drug
effect, and 100%
means all cells survived. Cell viability was calculated using the following
equation: Cell viability
(%) =(0D490nm of treatment/OD490nm of blank control) x 100%.
[0166] Western blot analysis. MCF-7/C6 cells were treated by different
conditions and then
collected by centrifugation at 14,000 rpm for 10 mM and lysed with a 1% (v/v)
Triton X-100
containing lysis buffer (50 mM Tris-HC1, pH 8.0, 150 mM NaCl) with protease
inhibitor.
Total cellular proteins were estimated using a BCA kit (Applygen). Each sample
(50 ug of
protein) was subjected to SDS-PAGE and transferred to nitrocellulose
membranes. After
blocking for 2 h at room temperature with 5% (wt/v) nonfat dry milk in blotto
solution (20 mM
Tris-HC1, pH 7.5, 150 mM NaCl and 0.1% Tween 20), the membranes were incubated
with
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primary antibody overnight at 4 C. Then the membranes were washed (3x5 min)
with
TBST solution and incubated with second antibodies for 2 h at room
temperature.
Signals were visualized by chemiluminescence on a Typhoon Trio Variable Mode
Imager.
Band density was calculated using NIH Image J software.
[0167] For HER2 dimer western blot analysis, MCF-7/C6 cells were treated with
indicated
protocols and then lysed in buffer containing 137 mM NaCl, 2.7 mM KC1, 10 mM
Na2HPO4, 1.8
mM KH2PO4, 1% Triton X-100 and protease inhibitors cocktail (Sigma-Aldrich).
The lysis
supernatant was collected after centrifugation at 12,000 rpm for 15 minutes.
0.2% glutaraldehyde
was added to the lysis supernatant for 10 minutes at 37 C. The lysis was
collected for western
blot analysis.
[0168] Animal model. All animal experiments were in accordance with protocols
No.19724,
which was approved by the Animal Use and Care Administrative Advisory
Committee at the
University of California, Davis. Female BALB/c nude mice were 6-8 weeks of age
(weight 22
2 g), which were purchased from Harlan (Livermore, CA, USA). MCF-7/C6 cells (5
x106 cells
per mouse) were inoculated subcutaneously into the flank of each female BALB/c
nude mice,
respectively. After around 10 days, NPs1-4 (8 mg/Kg) were injected via the
tail vein and ex vivo
images of -tumour, heart, liver, spleen, lung, kidney, intestine, muscle, skin
were collected at 10,
24, 48, 72, 168 h post injection. The images were collected by in vivo
fluorescence imaging
system (Carestream In-Vivo Imaging System FXPRO, USA). Tumour and Main organs
(heart,
liver, spleen, lung, kidney and brain) were collected and solidified with
glutaraldehyde (4%) at
72 h post injection of NPs for TEM imaging.
[0169] In vivo therapeutic effect. BALB/c nude mice with MCF-7/C6 cells (5 x
106 cells per
mouse) tumours inoculated subcutaneously into the flank were used in our
experiments. The
mice were randomly divided into five groups at 10 days post-tumour
inoculation. Each of them
treated with PBS, NPs1, NPs2, NPs3 and NPs4 every 48 h via i.v.
administration. During the
process of the treatment (40 days), the tumour volumes and body weight were
measured twice
per week. In parallel, the therapeutic effect of NPs1 was verified in the mice
bearing SKBR-3
and BT474 tumours with similar experimental method mentioned above. For
Haematoxylin and
eosin (H&E) staining test and Ki-67 test, MCF-7/C6 tumour-bearing mice were
sacrificed after
three times treatment and tumour tissues were collected.
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[0170] Statistical analysis. Data are presented as the mean standard
deviation (SD). The
comparison between groups was analyzed with the student's t-test (two-tailed).
One-way analysis
of variance (ANOVA) was used for multiple-group analysis. The level of
significance was
defined at *p < 0.05, **p < 0.01, and ***p < 0.001. All statistical tests were
two-sided.
Results and Discussion
[0171] Self-assembly and fibrillar-transformation of supramolecular material.
The
transformable peptide monomer 1 (TPM1'), BP-FFVLK-YCDGFYACYMDV, was prepared
with standard solid-phase peptide synthesis techniques followed by N-terminal
capping with bis-
pyrene, and its identity was confirmed by MALDI-TOF-MS (FIG. 6). For the
purpose of
comparisons, TPM2' (BP-GGAAK-YCDGFYACYMDV), TPM3' (BP-FFVLK-PEG1000) and
TPM4' (BP-GGAAK-PEG1000) were synthesized as negative controls (Table land
FIG. 7-9).
As the proportion of water in the mixed solvent (water and DMSO) of TPM1'
solution was
increased, there was a gradual decrease in absorption peaks (250-450 nm),
reflecting the gradual
formation of nanoparticles NPs1 via self-assembly, caused by 7C-7C interaction
and strong
hydrophobicity of BP and I3-sheet forming peptide sequence (FIG. 1A).
Concomitantly, the
fluorescence peak at 520 nm was found to increase dramatically, due to the ATE
fluorescence
properties of BP dye (FIG. 1B). TPM2', TPM3' and TPM4' all showed similar self-
assembling property. Nanoparticles (NPs1, NPs2, NPs3, and NPs4), assembled
from the four
TPMs by rapid aqueous dilution method, were analyzed by dynamic light
scattering (DLS) and
transmission electron microscopy (TEM) (FIG. 1C). The diameters of NPs1-4 were
found to be
around 20 nm, 30 nm, 25-60 nm and 20 nm, respectively.
Table 1. Molecular composition of transformable peptide monomers (TPMs) l'-4'
TPM BP FFVLK GGAAK YCDGFYACYMDV PEGi000
l' + + - + -
2' + - +
+ -
3' + + _
_ +
4' + - +
- +
TPM1 ' BP-FFVLK-YCDGFYACYMDV (with HER2 binding peptide, but without f3-sheet
forming peptide);
TPM2' BP-GGAAK-YCDGFYACYMDV (with HER2 binding peptide, but without f3-sheet
forming peptide);
TPM3' BP-FFVLK-PEG1000 (without HER2 binding peptide, but with f3-sheet
forming peptide);
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TPM4' BP-GGAAK-PEG1000 (without HER2 binding peptide nor f3-sheet forming
peptide).
[0172] To investigate the interaction of NPs1 with HER2 in vitro, soluble
extracellular
domain of HER2 protein as the transformation inducer was chosen. As shown by
the TEM
images in FIG. 1C, NPs1 was found to maintain a spherical structure at around
20 nm before
interaction with HER2. After incubation at room temperature with HER2 protein
for only 30 min
(molar ratio of HER2 peptide/HER2 protein z1000:1), a small number of
particulate nanofibrillar
structures (NFs1, width diameter about 10 nm) became apparent; more NFs1 were
detected at 6
h. By 24 h, a fibrillar network with a broad size distribution was clearly
detected, indicating that
the transformation process was receptor-mediated and time-dependent. No
transformation was
observed in the NPs1 preparation without the addition of HER2 protein, even
after 24 h. The
structural transformation from NPs1 to NFs1 was also confirmed in solution by
DLS (FIG. 1D),
with the gradual decrease in the 20 nm peak and corresponding increase in the
100 to 1000 nm
peak over time. In contrast, similar treatment of NPs2, NPs3 and NPs4
solutions with HER2 did
not reveal any significant changes over 24 h. Common features of the TPMs that
formed these
three negative control NPs were the lack of concurrent presence of the two
essential domains for
receptor-mediated transformation in NPs1: HER2 ligand and KLVFF I3-sheet
forming peptide.
Circular dichroism (CD) spectroscopy was used to monitor the conformation and
secondary
structure of TPM1' upon transformation (FIG. 1E). In the initial stage of
rapid self-assembly
to form NPs1, no obvious secondary structure was observed, probably because
hydrophobic
interactions induced by BP was too fast to form any intermolecular hydrogen
bonds. As NPs1
began to transform to NFs1 over the 24 h period in the presence of HER2, the
negative CD
signal at 216 nm and positive CD signal at 195 nm progressed gradually over
time, indicating [3-
sheet formation via hydrogen bonding. In addition to CD, the unique AIE
fluorescent property of
BP to monitor the kinetics of TPM1' transformation was exploited. As shown in
FIG. 1F, the
fluorescence intensity of BP in NPs1 dropped about 10% 30 min after addition
of HER2, but
turned around and increased as transformation to NFs1 progressed, and
eventually reached about
50% increase by 24 h. One plausible explanation for this interesting
observation is that the
packing density of BP or TPM1' in the fibrillar networks (NFs1 at 24 h) was
significantly higher
than that in the initial spherical structure (NPs1). However, during the
initial transformation
process when the spherical NPs1 were exposed to HER2, there was a transient
relaxation in the
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packing density prior to re-organization into the more densely packed nano-
fibrillar network. It is
also demonstrated that the particle size of NPs1 in PBS without HER2 remained
unchanged over
7 d at 37 C, whether 10% fetal bovine serum (FBS) was present or not.
[0173] The morphological characterizations of fibrillar-transformation of NPs.
To further
characterize the interactions between the transforming peptides and cell
surface receptors on
living cells, HER2+ breast cancer cell lines (SKBR-3 and BT474 cells) were
incubated with
NPs1, and then used confocal laser scanning microscopy (CLSM) to track the
fluorescent green
signal emitted by BP (FIG. 2A-2B). After 6 h incubation of NPs1 with these two
cell lines,
green fluorescence signal was observed on the cell surface rather than inside
the cells. In
contrast, for MCF-7 breast cancer cells with low-expression level of HER2, the
majority of the
fluorescent signal was found to reside inside the cells after 6-24 h (FIG.
2C), indicating that cell
surface display of HER2 protein was required for transformation of NPs1 to
nanofibrillar
network at the cell vicinity.
[0174] Radiotherapy is commonly used for the management of breast cancer
patients. It has
previously been reported that long-term fraction ionizing radiation (FIR) can
induce HER2
expression, both clinically and in experimental models. In fact, the HER2+ MCF-
7/C6 tumour
cell line used was derived from HER2 negative human breast cancer MCF-7 cell
line that had
undergone 30 days of FIR induction, followed by colony formation and clonal
isolation. MCF-
7/C6 cells exhibit the characteristic of radiation resistance, high expression
level of HER2, more
.. aggressive phenotype, and enhanced levels of cancer stem cell properties.
The relative expression
level of HER2 protein, determined by Western blot, was found to be 5 times
higher in MCF-
7/C6 cells than in MCF-7 cells (FIG. 2D). After 30 mm incubation of MCF-7/C6
cells with
NPs1 (100 pM), green fluorescent dots were observed on the cell membrane (FIG.
2E). By 24 h,
a luxuriant green fluorescent layer was found surrounding the entire cell.
[0175] To further validate the binding of NPs1 to HER2, rabbit anti-HER2
(29D8) monoclonal
antibody (MAb) was used to detect the extracellular domain of HER2 on the
surface of MCF-
7/C6 cells. Anti-HER2 MAb was labeled fluorescent red by the secondary Ab. The
NPs1 and the
transformed nanofibrillar network (NFs1) were labeled fluorescent green by the
intrinsic optical
property of BP. As shown in FIG. 2F, green fluorescence overlapped completely
with red
fluorescence around the periphery of the two cells. The merge image showed
overlapping green
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and red (to form yellow) around the cell surface, except the adhesion
interface between the two
cells, which was stained by just the anti-HER2 MAb (red fluorescence) and not
by the NPs 1.
This data was consistent with our notion that transformation of NPs1 to NFs1
was triggered by
its interaction with cell surface HER2 receptor exposed to the culture medium.
The cellular
distribution of negative control NPs (NPs2, NPs3 and NPs4) was also
investigated in MCF-
7/C6 cells. After 24 h incubation, the majority of the fluorescent signals
were found inside
the cells instead of on the cell surface . Scanning electron microscopy (SEM)
confirmed the
presence of nanofibrillar network (NFs1) on the surface of NPs1-treated MCF-
7/C6 cells but not
untreated cells (FIG. 2G). In contrast, no nanofibrillar structure was
detected on the surface of
cells treated with NPs2, NPs3 or NPs4 . Transmission electron microscopy (TEM)
were used
to better define the ultra-structure of the nanofibrillar network. Similar to
the result obtained
by SEM, abundant bundles of nanofibrils were detected on the surface of and in
between MCF-
7/C6 cells after incubation with NPs1 for 24 h. No nanofibrillar structure was
detected on
untreated MCF-7/C6 cells or cells treated with the three negative control NPs
for 24 h. In
another negative control experiment in which MCF-7, a cell line with low level
of HER2
expression, was incubated for 24 h with NPs1, only minimal amount of
nanofibrils were detected
on the cell membrane.
[0176] The extracellular and intracellular mechanisms of fibrillar-
transformation. It is
conceivable that HER2-mediated transformation of nanoparticle (NPs1) to
nanofibrillar network
(NFs1) could impair HER2 dimerization leading to suppression of downstream
signal
tranduction. To demonstrate this plausible mechanism, MCF-7/C6 cells were
incubated with
NPs1, NPs2, or PBS for 8 h (FIG. 3A). For the NPs1 treated cells, most of the
green
fluorescence signal (BP) was found to co-localize with the red fluorescence
(anti-HER2),
indicating that the nanofibrillar network was closely associated with HER2
receptors
displayed on the cell surface. For the cells treated with NPs2, in which the
HER2 ligand was
present but 3-sheet forming peptide was mutated, cell surface green
fluorescence was weak.
Furthermore, the green/red fluorescent signals on the membrane of the NPs1
treated cells
appeared to be significantly thicker and discontinuous, suggesting clustering
of nanofibrillar
structures and perhaps even disruption of cell membranes.
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[0177] The cytotoxic effect of NPs1 and the three negative control NPs on MCF-
7/C6 cells
after 48 h incubation was determined by a MTS assay. As shown in FIG. 3B,
treatment with
NPs1 resulted in significant cell death at a dose dependent manner, with cell
viability of 37% and
13% at 150 pM and 300 pM, respectively. Similar results were obtained for two
other HER2+
breast cancer cell lines, SKBR-3 and BT474. However, when MCF-7 cells with low
level of
HER2 expression were treated by these four NPs, no obvious cytotoxicity was
observed even at
the highest concentration of 300 M. This is consistent with our notion that
nanotransformation
and therefore cytotoxicity of NPs1 is HER2-mediated. To explore the mechanism
by which NPs1
induced apoptosis, the expression levels of various pro-apoptotic and anti-
apoptotic proteins were
evaluated by Western blot. As shown in FIG. 3C, treatment of MCF-7/C6 cells
with NPs1
resulted in down-regulation of anti-apoptotic protein Bc1-2 and up-regulation
of apoptotic protein
Bax, in a dose dependent manner. To study the effect of NPs1 on HER2
dimerization, a simple
method of brief chemical crosslinking with 0.2% glutaraldehye followed by
Western blot
analysis with anti-HER2 antibody was employed. This method has allowed us to
differentiate
dimeric HER2 from its monomeric form. It was clear from FIG. 3D and FIG. 3E
that NPs1
was able to inhibit HER2 dimerization in a dose-dependent manner. Time course
study indicated
that NPs1 (50 pM), not only could inhibit HER2 dimerization, it could also
promote conversion
of HER2 from dimeric form to monomeric form. The effect of NPs1 on MAPK
pathway was also
studied by Western blot. A significant decrease in pErk, pMek and pRaf-1 level
over time was
observed when the cells were treated with 50 laM of NPs1; this inhibitory
effect was dose-
dependent (FIG 3F). For the purpose of comparison, MCF-7/C6 cells were
incubated with 50
pIVI of each NPs for 36 h, and Herceptin was used as a positive control (FIG.
3G). Like
Herceptin, NPs1 was able to strongly inhibit phosphorylation of Erk, Mek and
Raf-1. In contrast,
the three negative control NPs did not significantly alter the phosphorylation
level of Erk, Mek
and Raf-1. Together, these data strongly support transformation of NPs1 to
nanofibrillar network
on the surface of HER2+ tumour cells causes inhibition of HER2 dimerization
and conversion of
HER2 dimers to monomers, leading to inhibition of downstream proliferation and
survival cell
signaling, and cell death.
[0178] In vivo evaluation of fibrillar-transformation. NPs1 was found to be
non-toxic;
blood counts, platelets, total protein, creatinine and liver function tests
obtained from normal
Balb/c mice treated with 8 consecutive q.o.d. doses of NPs1 were within normal
limit . For
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biodistribution studies, mice bearing MCF-7/C6 tumour were given i.v. NPs1;
10, 24, 48, 72 and
168 h later, main organs were collected for ex vivo fluorescent imaging study
(FIG. 4A-4B).
Fluorescent uptake by tumour and normal organs such as liver, lung and kidneys
were high at 10
h. Fluorescent signal persisted in tumour for over 3 days, with significant
residual signal even
after 7 days. In contrast, fluorescent signal in normal organs began to drop
after 10 h and was
almost undetectable in main organs at 72 h. At 72 h, tumour and overlying skin
were excised for
fluorescent microscopy studies. It was clear that compared to intense
fluorescent signal in
tumour, negligible signal was detected in the normal skin (FIG. 4C).
Histologic examination of
excised normal organs did not reveal any pathology. Similar in vivo
biodistribution studies on
.. NPs2, NPs3 and NPs4 were also performed in the same tumour model. At 72 h,
fluorescent
signal of -tumour derived from mice treated with NPs1 was found to be 2-3
times higher than that
of mice treated with NPs2-4 (FIG. 4D-4E). Prolonged retention of fluorescent
signal in NPs1
treated mice, even after 7 days, could be attributed to in situ receptor-
mediated transformation of
NPs1 into NFs1 networks at the tumour microenvironment. TEM studies on excised
tumour, 72 h
after i.v. administration, showed abundant bundles of nanofibrils in the
extracellular matrix of
tumour sections. No such nanofibrils were observed in the negative control NP-
treated and
untreated mice (FIG. 4F). In addition, many cells in the tumour excised from
NPs 1-treated
mouse appeared to be dying with large intercellular spaces. The TEM images of
other organs
(heart, liver, spleen, lung, kidney and brain), excised from the same mouse
were found to be
normal, without any sign of nanofibrillar networks, which was consistent with
the result of the
optical imaging and histopathology studies mentioned above.
[0179] Anti-tumour activity of fibrillar transformable NPs. Therapeutic
efficacy studies of
NPs1, NPs2, NPs3 and NPs4 were performed in MCF-7/C6 HER2+ breast cancer
bearing mice
(FIG. 5A). When tumour volume of mice reached about 50-80 mm3, NPs were
injected
.. consecutively 8 times q.o.d. (day 1, 3, 5, 7, 9, 11, 13, 15) via tail vein
and observed continuously
for 40 days. As shown in FIG. 5B, tumour volume of NPs1 treated mice gradually
shrunk and
was totally eliminated after treatment without any sign of recurrence. In
contrast, none of the
other 3 negative control groups (NPs2, NPs3, and NPs4) elicited any
significant tumour
response. None of the mice in this therapeutic study showed any symptoms of
dehydration and
significant body weight loss during the entire 40 d therapeutic study (FIG.
5C). The survival
curves correlated well with tumour growth results (FIG. 5D). Seven of the
eight mice receiving
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NPs1 treatment survived over 150 days without any sign of tumour recurrence.
One of these
eight mice, no longer with detectable tumour, died at around day 60 for
unknown reason. In
contrast, all mice in the PBS, NPs2, NPs3 and NPs4 treated groups died within
51, 63, 57, and 60
days respectively. This result is highly encouraging and clearly demonstrates
the clinical
potential of receptor-mediated transformative supramolecular nanotherapeutics
(e.g. NPs1)
against solid tumour in general, and more specifically against HER2+ tumours.
[0180] To better understand the in vivo anti-tumour mechanism of NPs1, mice
were
sacrificed and residual tumours collected for biochemical and morphological
assessment after
3 consecutive q.o.d injections of NPs1 (FIG. 5E). Frozen sections were
obtained for fluorescent
microscopy and hematoxylin and eosin (H&E) stain (FIG. 5F). The degree of cell
kill was found
to correlate well with that of fluorescent intensity; necrosis was detected in
the tumour areas with
strong fluorescence intensity. To understand how the nanofibrillar network
kill the HER2+
tumour cells, high magnification TEM on tumours obtained from NPs1-treated
mice was
performed. The TEM image of a necrotic or necroptotic cell in FIG. 5G revealed
that the
plasma membrane was broken, with abundant fibrillar nanostructures present
inside the broken
cell. Some of the nanofibrillar bundles were found adjacent to the nuclear
envelope of the
nucleus. No significant cell kill was detected in tumour sections obtained
from mice treated with
PBS, NPs2, NPs3 or NPs4. Tissue section staining for Ki-67 marker is a good
way to assess the
anti-proliferative effects of NPs1 in vivo. After 3 treatments with NPs1, the
expression level of
Ki-67 in tumour tissue was markedly decrease, compared to the tumour obtained
from mice
treated with negative control NPs (FIG. 51I).
[0181] It has been shown above that NPs1 could inhibit HER2 dimerization and
phosphorylation of Erk, Mek and Raf-1 in HER2+ cell line in cell culture. Here
similar Western
blot studies are performed on tumours excised from mice that had undergone 3
consecutives
q.o.d. treatments of NPs1. As shown in FIG. 51, total HER2 level remained
unchanged, but
phosphorylation of Erk, Mek and Raf-1 was found to be markedly decrease,
compared to the
other negative control groups. Together, the data clearly demonstrated that
receptor-mediated
transformative supramolecular nanotherapeutic NPs1 was highly effective in
suppressing
downstream proliferative and survival cell signaling at the tumour tissue
level. To better
investigate the universality of NPs1 as an efficacious therapeutic against
HER2+ tumours, two
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other human HER2+ breast cancer xenograft models (SKBR-3 and BT474) were
chosen for our
studies. As shown in FIG. 5J-5K, the tumour volume of mice treated with NPs1
responded very
well with complete elimination of SKBR-3 tumour, and almost completed
elimination of BT474
tumour by day 40. In contrast, the tumour volumes of the PBS control groups
had grown to
1200-1500 mm3 on day 40.
[0182] One known side-effect of Herceptin is cardiotoxicity. It cannot be
given to patient
together with cardiotoxic drug such as doxorubicin. Thus far, there was no
observed cardiotoxic
effects in our xenograft studies with NPs 1. No uptake of NPs1 in the
myocardium was detected.
This is not surprising as the coronary vessels are expected to be intact and
the 20 nm NPs1 will
not be able to reach the myocardium. The fact that NPs1 was highly efficacious
against three
different HER2+ tumours warrants further preclinical and clinical development
of NPs1 against
HER2+ breast, ovarian, gastric, and bladder cancers. There is good clinical
evidence that some
originally HER2 negative breast cancers can be induced to express HER2 after
long-term
fraction ionizing radiation (FIR). This further expands the patient population
who may benefit
from this novel receptor-mediated transformable nanotherapy (RMTN).
[0183] It has been demonstrated that 8 consecutive q.o.d doses of NPs1 alone
as a
monotherapy was efficacious in curing a large percentage of mice bearing
relatively small (<100
mm3) HER2+ breast cancer xenografts.
Example 2: Nanocarriers comprising a plurality of two different conjugate
[0184] Immune checkpoint blockade (ICB) therapy has revolutionized clinical
oncology. One
of the main contributing factors for ICB resistance is defects in Teff cell
homing to the tumour
sites. This example describes a 28 nm non-toxic peptidic micellar
nanoparticle, displaying
LXY30, an a331 integrin targeting ligand. Upon interaction with a331 integrin
over-expressed in
many epithelial cancers, these nanoparticles would undergo in situ
transformation at the tumour
microenvironment (TME) into nanofibrillar structural network. The
nanofibrillar network not
only promotes cytotoxic CD8+ T cell homing to and macrophage re-education at
the tumour
sites, but also allowed sustain release of TLR 7/8 immunoagonist (resiquimod),
via esterase at
the TME, resulting in elimination of syngeneic 4T1 breast cancer and Lewis
lung cancer models
in mice, when given together with anti-PD-1 antibody. These structural
transformation-based
supramolecular peptides represent an innovative class of receptor-mediated
targeted
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immunotherapeutics against cancer via enhancing T cell tumour homing and
reprogramming of
TME.
[0185] This example describes a ligand-receptor-mediated, peptide-based, and
non-toxic dual-
ligands fibrillar transformable nanoplatform, capable of mounting systemic
anti-immune
response against cancers. This nanoplatform, initially in nanoparticle form,
is self-assembled
from two smart transformable peptide monomers TPM1 and TPM2. TPM1, LXY30-
KLVFFK(Pa), was comprised of three discrete functional domains: (1) the high-
affinity and
high-specificity LXY30 cyclic peptide (cdG-Phe(3,5-diF)-G-Hyp-NcR) ligand that
targets a3[31
integrin heterodimeric transmembrane receptor expressed by many solid tumours,
(2) the
KLVFF 13-sheet forming peptide domain originated from 13-amyloid (A13)
peptide, and (3) the
pheophorbide a (Pa) moiety with fluorescence property, serving as a
hydrophobic core to induce
the formation of micellar nanoparticles. TPM2,proLLP2A-KLVFFK(R848), was also
comprised
of three discrete functional domains: (1) proLLP2A, the "pro-ligand" version
of LLP2A, which
is a high-affinity and high-specificity peptidomimetic ligand against
activated a4131 integrin of
lymphocytes, (2) the same KLVFF 13-sheet forming peptide domain, and (3) R848
(resiquimod),
a hydrophobic toll-like receptors (TLRs) 7/8 agonist, grafted to TPM2 main
chain via an ester-
bond. In proLLP2A, the carboxyl group of LLP2A is esterized by 3-methoxy-1-
propanol such
that it will not interact with normal lymphocytes and mesenchymal stem cells
during blood
circulation. At the TME with abundant esterase, proLLP2A will be converted to
LLP2A to
facilitate homing of immune cells to the tumour sites. Similarly, esterase-
responsive release of
R848 would occur at the TME to activate antigen-presenting cells (APCs),
promote immune
cells to produce anti-tumour response factors, and reverse the phenotype of
macrophage from
M2 to Ml.
[0186] Under aqueous condition and in blood circulation, TPM1 and TPM2 would
self-
assemble into one spherical transformable nanoparticle (T-NP) at a ratio of
1:1, in which
KLVFFK(Pa) and KLVFFK(R848) domains constituted the hydrophobic core, and
LXY30 and
proLLP2A ligand peptides constituted the hydrophilic corona. Upon interaction
with (13(31
integrin receptor protein displayed on the tumour cell membrane, the T-NPs
would undergo in
situ transformation into nanofibrillar (T-NFs) structural network on the
surface of tumour cells
and within the TME where the tumour associated exosomes were abundant, thus
maintaining a
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prolonged retention of the nanofibrillar network at the tumour sites (at least
7 days). In this case,
the more hydrophilic proLLP2A peptide ligand would be displayed on the outer
surface of the
fibrils, while the hydrophobic Pa and R848 would be sequestered at the core of
the fibrils. With
the elevated esterase in the TME and on the tumour cells, proLLP2A would
quickly be converted
to LLP2A (T cell ligand) against activated a4431 integrin. LLP2A displayed on
the fibrils would
facilitate the homing and retention of activated immune cells such as Teff
cells (e.g. CD8+ T) cells
at the TME and adjacent to the tumour cells. It would also enhance the
interaction between T cell
receptor (TCR) of Teff and major histocompatibility complex (MHC) of tumour
cells. The
addition of anti-PD-1 ICB therapy would further enhance the anti-tumour immune
response by
activating the cytotoxic T cell and reversing the dysfunction and exhaustion
of Teff. In addition,
the sustained release of R848 from the nanofibrillar network as a result of
the elevated esterase at
the tumour site would reverse the immunosuppressive TME. These structural
transformation-
based supramolecular peptides represent an innovative class of receptor-
mediated targeted
immunotherapeutics against cancer via enhancing T cell homing to the tumours
and improving
the TME from an immunosuppressive state to a durable immunoactive state (FIG.
12).
[0187] Self-assembly and fibrillar transformation of the nanoplatform. Two
transformable
peptide monomers (TPM1: LXY30-KLVFFK(Pa); TPM2: proLLP2A-KLVFFK(R848)) were
synthesized and characterized (FIG. 13A and FIG. 20). As the proportion of
water in the mixed
solvent (water and DMSO) of the TPM1 and TPM2 mixture solution (the ratio of
1:1) was
increased, there was a gradual decrease in fluorescence peak at 675 nm due to
the ACQ
properties of Pa dye (FIG. 13B), reflecting the gradual formation of
transformable NPs (named
as T-NPs) via self-assembly. Concomitantly, there was a modest decrease in the
absorption peak
at both 405 and 680 nm. Nanoparticles were analyzed by transmission electron
microscopy
(TEM) and dynamic light scattering (DLS). TPM1 and TPM2 each alone were able
to self-
assemble to form spherical nanoparticles (NPsTpmi and NPsTpm2) at 18 and 55
nm, respectively.
T-NPs, assembled from 1:1 mix of TPM1 and TPM2, yielded a spherical structure
at around 28
nm, which fell between the sizes of NPsrpmi and NP5TPM2 (FIG. 21A). The
critical aggregation
concentrations (CAC) of T-NPs was determined to be 8 laM (FIG. 21B). It was
also
demonstrated that T-NPs could maintain good serum stability and proteolytic
stability over 7
days at 37 C (FIG. 21C).
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[0188] To verify the receptor-mediated fibrillar transformable process of T-
NPs in vitro,
soluble a3[31 integrin protein (receptor for LXY30) was added to T-NPs
solution. After 24 h of
incubation at room temperature, a fibrillar network (T-NFs, width diameter
about 8 nm) with a
broad size distribution was clearly detected (FIGs. 13C, 13F). No
transformation was observed
in the T-NPs preparation without the addition of a3131 integrin protein, even
after 24 h (FIG.
21D). The CAC of T-NFs was determined to be 5 laM, which was lower than that
of T-NPs (8
laM), indicating that T-NFs has higher propensity to form nanostructures than
T-NPs (FIG.
21E). The fluorescence of Pa was also used to monitor the fibrillar-
transformation process of T-
NPs (FIG. 13D). Addition of a3(31 integrin protein to T-NPs solution resulted
in a gradual
decrease in the fluorescence intensity of Pa, and a remarkable shift of the
fluorescent peak
towards the red region from 680 nm to 725 nm within the first 2 h, consistent
with the change in
aggregation structure of Pa from spherical structure to fibrillar
configuration during that time.
Responsiveness ofproLLP2A and LLP2A displayed on the T-NPs surface to soluble
(1.4131
integrin protein in the presence and absence of esterase was
investigated(FIGs. 13E-13F).
Soluble (1.4131 integrin protein alone was not able to alter the spherical
structure of T-NPs
displaying proLLP2A, even after 24 h of incubation. In contrast, successive
addition of esterase,
followed by soluble (1.4131 integrin protein was able to elicit conversion of
spherical T-NPs to
fibrillar network after 24 h of incubation. This result confirmed that
esterase was able to convert
pro-ligand proLLP2A to ligand LLP2A, which in turn was able to trigger
receptor-mediated
transformation of T-NPs to T-NFs. Circular dichroism (CD) spectroscopic
analysis of the
transformation process of T-NPs showed a gradual progression of a negative
signal at 216 nm
and a positive signal at 195 nm upon incubation with a3(31 integrin protein or
combination
esterase/a431 integrin protein, indicative of (3-sheet formation (FIG. 2G) and
consistent with
TEM results shown in FIGs. 13C and 13E. In vitro release behaviour of R848
from T-NFs was
studied at pH 6.5 with addition of esterase to simulate TME condition. As
shown in FIG. 1311,
about 45% of R848 was released the first 24 h, after which the release rate
gradually slowed
down and about 86% cumulative release was observed by 168 h, indicating that
prolonged and
sustained release of R848 could occur at the TME. To demonstrate the unique
transformable
property of T-NPs, a related control untransformable nanoparticle (UT-NP) was
formed by
assembly of two TPMs without 13-sheet forming KLVFF peptide sequence, at a
ratio of 1:1
(TPM3: LXY30-KAAGGK(Pa) and TPM4: proLLP2A-KAAGGK(R848)). As expected, a313.1
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integrin protein was unable to transform UT-NPs to fibrillar structures even
after 24 h, indicating
that the 13-sheet peptide was required for the transformation of T-NPs to T-
NFs (FIGs. 20 and
21).
[0189] In vitro evaluation of fibrillar-transformation of nanoparticles and T
effector cell
homing to tumour sites. To further characterize the interaction between
transformable
nanoparticles and a3131 integrin receptors on the surface of living cells,
a3131 integrin expressing
4T1 murine breast cancer cell was chosen. Flow cytometry analysis confirmed
that LXY30, the
high affinity a3131 integrin ligand, did bind to 4T1 tumour cells (FIG. 23).
It was also found that
T-NPs was slightly cytotoxic against 4T1 cells, with 85% cell viability at 50
uM (FIG. 24). The
.. distribution of NPs was investigated by tracking the red fluorescent signal
emitted by Pa using
confocal laser scanning microscopy (CLSM). Six hours after incubation of 4T1
cells with T-NPs,
a strong red fluorescence signal was observed on the cell surface and its
vicinity but not inside
the cells (FIG. 15A). In contrast, the fluorescent signal of Pa in UT-NPs-
treated group was
found to be concentrated primarily in the cytoplasm of the cells. To study the
retention and
.. stability of formed nanofibrillar network on the surface of tumour cells,
unbound NPs were
washed off after 6 h of incubation and fresh medium without NPs was added to
incubate cells for
another 18 h. T-NPs treated cells still retained strong red fluorescence
signals on the cell surface
at 24 h (FIG. 15B). In sham contrast, only weak fluorescence signal was
observed inside the
cells treated with UT-NPs after 24 h. This is probably due to the enzymatic
degradation of the
.. already endocytosed UT-NPs after 18 h of incubation, but without any new
endocytic uptake
during that time period. TEM images confirmed the presence of nanofibrillar
network (T-NFs)
on the surface of, and between 4T1 cells after incubation with T-NPs for 24 h,
but absence of
such nanofibrillar structures on cells treated with UT-NPs (FIG. 15C). The
fibrillar structures
further away from the cell surface were probably induced by the secreted
tumour exosomes
displaying (13(31 integrin proteins. The effect of esterase on the
interactions between T-NPs and T-
cell surface a4131 integrin, after converting pro-ligand pro-LLP2A to LLP2A
displayed on the
surface of T-NPs was investigated. Live GFP transfected Jurkat T-lymphoid
leukemia cells with
high expression level of constitutively activated a4131 integrin protein were
used to mimic T cells.
As shown in FIG. 15D, after 6 h incubation of Jurkat cells with T-NPs (pre-
treated with
esterase), a luxuriant red fluorescent layer was found surrounding the Jurkat
cells, indicating that
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the conversion of pro-ligand to LLP2A ligand was successful. Scanning electron
microscopy
(SEM) confirmed the presence of fibrillar network on the surface of T-NPs-
treated 4T1 cells and
esterase pre-treated T-NPs-treated Jurkat cells (FIG. 15E).
[0190] To simulate the processes of initial fibrillar transformation of T-NPs
on the 4T1 cells
surface followed by T cell homing, first incubated 4T1 cells with T-NPs for 6
h, unbound T-NPs
were then washed off, followed by addition of fresh medium containing esterase
but without T-
NPs. After 1 h of incubation, Jurkat cells were added and incubated with 4T1
cells for 2 or 4 h.
After that, unbound Jurkat cells were gently removed prior to CLSM imaging
(FIG. 15F). As
expected, a fibrillar structure layer with red fluorescence was detected
surrounding 4T1 cells
surface, and Jurkat cells (GFP+) were found to interact with the red
fluorescent fibrillar network
and in close proximity to 4T1 breast tumour cells, after 2 h of incubation. As
the incubation time
was increased to 4 h, many more Jurkat cells were found clustered around the
4T1 tumour cells,
which was consistent with our notion that fibrillar network would facilitate
the homing of
immune cells such as T-cells to the tumour sites. SEM imaging provided
critical evidences that
the nanofibrillar structures had played a significant role in direct physical
contact between 4T1
cells and Jurkat cells through nanofibrillar network (FIG. 15G).
[0191] The conversion of TAMs from an immunosuppressive M2-polarized phenotype
to an
anti-tumourigenic Ml-polarized phenotype is one of the major immunotherapeutic
strategies for
reversing the immunosuppressive tumour microenvironment. Macrophage
polarization states
demonstrate hallmark morphology, e.g., elongated projections for M2-like cells
as opposed to a
round and flattened morphology for Ml-like counterparts. IL-4 has been used to
induce bone
marrow derived macrophages (BMDM) to M2-polarized macrophages, as reflected by
the
increase in expression level of the metabolic checkpoint enzyme arginase-1
(Argl) and mannose
receptor-1 (Mrcl). R848 has been reported to be a powerful driver of the Ml-
phenotypes in
vitro, resulting in elevated level of interleukin 12 (IL-12) and nitric oxide
synthase (Nos2)
produced by these cells. The possibility of using T-NFs to re-educate
macrophages from M2
phenotype to M1 phenotype was investigated. In the nanoplatform, R848 was
covalently linked
to TMP2 via an ester bond. Therefore, not unexpected, incubation of 4T1 cells
with T-NFs,
preformed from T-NPs with soluble a3r31 integrin protein, did not have
significant effect on M2-
polarized macrophages induced by IL-4 (FIG. 1511). No significant change in
macrophage
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morphology and expression level of Argl and Mrcl was observed even after 12 h,
which can be
explained by the lack of R848 released from T-NFs. In contrast, addition of
esterase to the
culture medium followed by 12 h incubation resulted in morphological change of
M2-state
macrophages towards Ml-state, a decrease in Argl and Mrcl, and an increase in
IL-12 and Nos2
expression as measured by qPCR. These changes were even more pronounced after
24 h, at
which time the macrophages were completely transformed to a round and
flattened morphology
(Ml-like), with further decrease in Argl and Mrcl, and increase in IL-12 and
Nos2 expression.
The ability of T-NFs to anchor at the TME, afforded the sustained release of
R848 from the
fibrillar network, will generate a durable anti-cancer immunoactive TME.
[0192] In vivo evaluation of fibrillar-transformation of nanoparticles and
tumour homing
of T effector cells. T-NPs was found to be non-toxic: blood counts, platelets,
creatinine and
liver function tests obtained from normal Balb/c mice treated with eight
consecutive q.o.d.
intravenous (i.v.) doses of T-NPs were within normal limits (FIGs. 25-26). In
vivo blood
pharmacokinetics (PK) studies indicated that T-NPs possessed a long
circulation time (T-half
(a): 2.866 h and T-half (3): 23.186 h), indicating its stability during
circulation (FIG. 27). For
biodistribution studies, T-NPs were tail vein injected into Balb/c mice
bearing syngeneic
orthotopic 4T1 breast cancer; 10, 24, 48, 72, 120 and 168 h later, tumour and
main organs were
excised for ex vivo fluorescent imaging (FIG. 16A-16B). Significant
fluorescent signal of Pa
was found to persist in tumour tissue for over 168 h, while fluorescent signal
in normal organs
began to decline after 10 h and was almost undetectable in the main organs at
72 h. In sharp
contrast, fluorescent signal of Pa at tumour tissue treated by UT-NPs was
found to gradually
decline over time after peaking at 24 h (FIG. 16C-16D). By 168 h, less than
2.88% of the peak
fluorescent signal for UT-NPs remained in the tumour, whereas for T-NPs, over
59.89% signal
remained in the tumour (FIG. 16D). Prolonged retention of fluorescent signal
in T-NPs-treated
mice could be attributed to in situ receptor-mediated transformation of T-NPs
into T-NFs
networks in the TME. TEM studies on excised tumour sections, 72 h after i.v.
administration,
showed abundant bundles of nanofibrils in the extracellular matrix while no
such nanofibrils
were observed in negative control UT-NPs-treated and saline-treated mice (FIG.
16E).
Fluorescent micrographs of tumour and overlying skin revealed intense
fluorescent signal in
tumour region but negligible signal in normal skin. This is consistent with
our notion that (1) T-
NPs would leak into the TME through leaky tumour vasculatures (EPR effect),
followed by
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interaction with a3131 integrin on tumour cells and tumour associated exosomes
to generate T-
NFs, and (2) blood vessels are not leaky in normal skin (FIG. 16F). The tissue
distribution of
R848 over time was also determined with high pressure liquid chromatography-
mass
spectroscopy (HPLC-MS). It was found that with T-NPs, R848 uptake by tumour
was
significantly higher than that of other normal organs at 24 h, and that the
retention of R848 at
tumour site was quite high at 1.18 pg per g tissue even at 7 days after
injection (FIG. 16G).
Although UT-NPs could also deliver significant amount of R848 to the tumour
site (80% of what
T-NPs could deliver), but retention of R848 at the tumour site was much lower
than that of T-
NPs. Prolonged retention of R848 in tumour site indicates that a sustained
immune-active TME
.. could be achieved with T-NPs.
[0193] To evaluate if the nanofibrillar networks displaying LLP2A and R848 at
the TME
could promote in vivo T cells homing to the tumour sites, tumours from T-NPs-
treated mice were
excised on day 15 after a single i.v. injection of T-NPs, and the immune cell
populations within
the tumours were analyzed by flow cytometry, immunohistochemistry (IHC) and
qPCR.
Experiment using UT-NPs as an untransformable/endocytic negative control group
was also
performed at the same time. It was found that tail-vein injection of T-NPs had
resulted in a
sustained immunoactive TME. First, T-NPs was found to significantly stimulate
the production
of chemokine CXCL10 at the tumour site (FIG. 1611), which was known to
facilitate T effector
cells recruitment. It was observed that the proportion of CD45+CD3+ and
CD45+CD3+CD8+ T
cells in the T-NPs-treated tumour tissue was substantially higher than those
from mice treated
with endocytic UT-NPs or saline alone (FIG. 16I-16J). More specifically, the
percentage of
CD3+CD8+ T effector cells in tumours was found to be 18 and 4-fold increase,
relative to that of
saline and UT-NPs-treated mice, respectively (FIG. 16J). Second, it was found
that the relative
abundance of CD4+Foxp3+ Tregs at the tumour site was substantially lower in
mice that received
T-NPs treatment than those in mice treated with UT-NPs, i.e. (4.97% versus
13.0%) or saline
(4.97% versus 14.6%) (FIG. 16K). The ratios of tumour-infiltrating CD8+ killer
T cells to
immunosuppressive Tregs (CD3+CD4+Foxp3+), which could be an indicator of anti-
tumour
immune balance, were found to be the highest in T-NPs treated group (FIG. 16J-
16K). IHC
staining of tumour tissue sections also confirmed an increase in CD8/CD4 and
decrease in Foxp3
(FIG. 16L). Third, IHC staining of tumour sections demonstrated an increase in
Ml-polarized
macrophage marker CD68 and a decrease in M2-polarized macrophage marker CD163
in the T-
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NPs treated group, compared to the tumour tissue treated by UT-NPs. This could
be explained by
the sustained release of R848 at the tumour site, causing the phenotypic re-
education of TAMs.
Fourth, gene expression level of cellular immune related markers (IFN-y, TGF-
13) and
macrophage markers (IL-12, IL-10, Nos2 and Arg-1) were also evaluated by qPCR.
As shown in
FIG. 16M, the high expression level of IFN-y and low expression level of TGF-
13 in the tumour
tissue confirmed that a strong tumour-specific immune response had been
elicited. Furthermore,
the secretion of IL-12 and Nos2 was found to be significantly upregulated,
while the secretion of
IL-10 and Arg-1 was significantly down-regulated, indicating a significant
phenotypic
conversion of TAMs from M2 state to M1 state, with T-NPs treatment, but not UT-
NPs
treatment nor saline control.
[0194] Therapeutic efficacy study was performed in syngeneic orthotopic 4T1
breast cancer-
bearing mice. Mice were randomly divided into six groups, each received a
different treatment
regimen: (1) Saline; (2) (EK)3-KLVFFK(Pa)/(EK)3-KLVFFK(R848); (3) proLLP2A-
KLVFFK(R848) (single monomer); (4) LXY30-KAAGGK(Pa)/proLLP2A-KAAGGK(R848)
(untransformable UT-NPs); (5) LXY30-KLVFFK(Pa)/proLLP2A-KLVFFK(Pa) (fibrillar-
transformation but absence of R848); (6) LXY30-KLVFFK(Pa)/proLLP2A-
KLVFFK(R848).
Regimen 6 is the complete T-NPs, containing all 4 critical components:
LXY30,proLLP2A,
R848, and KLVFF, whereas regimen 2, 3, 4 or 5 all lack some components of T-
NPs. When
tumour volume reached about 50 mm3, all treatment regimens were tail vein
injected
consecutively eight times q.o.d. and the mice were continuously observed for
21 days (FIG.
17A). As shown in FIG 17B, regimen 2, 3 and 4 were inactive. Regimen 5
(fibrillar-
transformation but no R848) demonstrated significant tumour suppression
compared to group 2,
3 and 4. Regimen 6 (T-NPs, both fibrillar-transformation and R848) was found
to be the most
efficacious with significant tumour growth suppression (FIG. 17B) and
prolonged survival
(FIG. 17D), indicating the importance of combination T cells homing strategy
and sustained
release of TLR7/8 agonist. None of the mice in this therapeutic study showed
any symptoms of
dehydration nor significant body weight loss during the entire treatment
period (FIG. 17C). The
survival curves correlated well with tumour growth results. The mice treated
by regimen 6 (or T-
NPs) achieved a longer median survival time (62 d) compared with other
treatment groups (29,
32.5, 33.5, 33.5 and 39 d for regimen 1, 2, 3, 4, and 5, respectively).
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[0195] To elucidate the mechanism of immunotherapeutic effects induced by
transformable
nanoparticles, the tumour tissues were collected and used flow cytometry to
quantify tumour-
infiltrating CD3+ (CD45+CD3+) and CD8+ (CD45+CD3+CD8+) T cells (FIG. 17E).
Only the
treatment regimen capable of in situ fibrillar transformation and presentation
ofproLLP2A
(regimen 5 and 6) significantly increased the frequency of CD3+ and CD8+ T
cells within the
tumours, particularly in combination with immune adjuvant R848 in T-NPs
(regimen 6), which
was consistent with the observed strongest anti-tumour effects in T-NPs.
Tumour sections
(H&E) obtained from mice treated with T-NPs revealed a marked decrease in Ki-
67 expression,
an increase in CD8+ T cells, and a decrease in Foxp3 (Treg cells), compared
with other control
groups (FIG. 17F). There was an increase in CD68 and a decrease in CD163,
indicating that the
phenotype of macrophages was reversed after 8 doses of T-NPs. It is known that
CD8+ T cells
secrete cytokines IFN-y and TNF-a to kill tumour cells. The expression levels
of IFN-y and
TNF-a in the tumour tissue were further evaluated by qPCR. As shown in FIG.
17G, treatment
regimen 6 (T-NPs) was the most efficacious in restoring the immunoactive state
of the tumour
microenvironment, with the highest expression levels of IFN-y and TNF-a. In
addition, T-NPs
also significantly induced expression of IL-12, IL-6 and Nos2, and suppressed
expression of
TGF-13, IL-10 and Arg-1, leading to the suppression of the Treg cells
recruitment and re-
education of M2-like macrophages to M1 phenotype.
[0196] Although promising, T-NPs alone, however, was not able to completely
eliminate the
tumour. This may be caused by insufficient activation and homing of T effector
cells in the
tumour microenvironment. It is well known that tumour cells hijack PD-1
receptors of T cells by
overexpression of PD-L1, which can activate PD-1, leading to inhibition of T
cell proliferation,
activation, cytokine production, altered metabolism and cytotoxic T
lymphocytes killer
functions, and eventual death of activated T cells. Clinically, antibodies
targeting PD-1 or PD-Li
have been demonstrated to be able to reinvigorate the "exhausted" T cells in
the tumour
microenvironment. However, except for melanoma and non-small cell lung cancer,
the clinical
response rate of ICB anti-PD-1 or anti-PD-Li therapy is limited and most
patients are still
refractory. One critical reason is that there are not enough Teff cells in the
tumour
microenvironment. Our receptor-mediated fibrillar transformable nanoplatform
(promoting T
cells homing and improving tumour microenvironment) may be able to correct
such deficiency,
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and therefore will greatly synergize PD-1 and PD-Li checkpoint blockade
immunotherapy.
Syngeneic orthotopic 4T1 breast cancer-bearing mice were randomized into four
groups for anti-
PD-1 antibody (anti-PD-1) therapy with or without additional nanoplatform: (1)
anti-PD-1 alone;
(2) regimen 4 (UT-NPs) plus anti-PD-1; (3) regimen 5 plus anti-PD-1; (4)
regimen 6 (T-NPs)
plus anti-PD-1. When tumour volume reached about 100 mm3, NPs were given i.v.
injected on
day 1, and anti-PD-1 given i.p. on day 2. The same cycle was repeated on day
3, 5, 7, and 9 for a
total of 5 cycles, and mice were observed continuously for 21 days (FIG. 18A).
Not
unexpectedly, anti-PD-1 alone and regimen 4 plus anti-PD-1 treatment were
ineffective (FIG.
18B). In contrast, regimen 5 plus anti-PD-1 treatment did significantly
suppress tumour growth,
resulting in a longer median survival, compared with 8 treatments of regimen 5
without anti-PD-
1 as shown in FIG. 18B,18D (49.5 d vs. 39 d); both of these treatments however
were not able to
completely eliminate the tumours. Most remarkably, mice treated with regimen 6
(T-NPs) plus
anti-PD-1 resulted in gradual shrinkage and eventual complete elimination of
tumours within 21
days, and without any sign of recurrence during the observation period of 90
days (FIG. 18C),
validating the synergistic effects of our transformable nano-immuno-platform T-
NPs with
checkpoint blockade immunotherapy.
[0197] Unlike traditional chemotherapy or targeted therapy in clinical
oncology,
immunotherapy can potentially induce an adaptive response with capacity for
memory. Memory
is crucial to achieving durable tumour responses and preventing recurrence,
which often leads to
mortality. To assess whether the synergistic therapy of T-NPs with immune
checkpoint anti-PD-
1 therapy (T-NPs plus anti-PD-1 Ab) could induce a memory response, the cured
mice from
previous experiment was re-challenged (FIG. 18A-18C) with 4T1 cells on the
opposite
mammary fat pad on day 90; naive mice of the same age were used as a negative
control (FIG.
18D). In this experiment, the mice were given anti-PD-1 via i.p. three times
on day 91, 93 and
95. The tumour volume of all the naive mice increased rapidly within 30 days
even with the
injection of anti-PD-1 (FIG. 18E). However, either no tumour growth or
significant delay in
tumour growth was observed in mice previously treated successfully with T-NPs
plus anti-PD-1
treatment (FIG. 18F), confirming the presence of an excellent immune memory
response exerted
by these previously treated mice. Survival curves of this experimental group
correlated well with
tumour growth results (FIG. 18G). All mice remained alive during the 60-day
observation
period (day 90-150). In addition, the serum levels of cytokines such as TNF-a
and IFN-y in this
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experimental group were found to be much higher than those in the control same
age naive mice
group after re-challenged with 4T1 tumour cells for 6 days (FIG. 1811-184
These results
suggest that a durable and robust T cell memory response was generated by
regimen 6 (or T-
NPs) plus anti-PD-1 given previously.
[0198] In addition to 4T1 syngeneic orthotopic breast cancer model, similar
therapeutic study
in Lewis lung syngeneic subcutaneous murine tumour model was performed with
excellent
results (FIG. 18J-18L). Complete tumour regression and prolonged survival was
obtained for
therapy with T-NPs plus anti-PD-1. No systemic toxicity and weight loss were
detected.
[0199] In spite of the clinical success of checkpoint blockade immunotherapy,
only a fraction
of cancer patients benefits from this therapy. Defects in Teff cells homing to
the tumour sites is
probably one the main reasons why many patients remain refractory to such
treatment.
Development of approaches to convert an immunologically "cold" tumour to a
"hot" tumour is
undergoing intense investigation around the world. The receptor-mediated
transformable
nanoparticles (T-NPs) described herein can provide a relatively simple
solution to this challenge.
By incorporating pro-ligand LLP2A and R848 to the nanoparticle, it has been
demonstrated in
syngeneic 4T1 breast cancer and Lewis lung cancer model that this non-toxic
treatment can (1)
facilitate the homing of T-cells to the tumour sites, (2) promote retention of
T-cells at close
proximity to the tumour cells, and (3) provide sustained release of R848 at
the tumour
microenvironment, resulting in the re-education of TAMs to M1 phenotype. Since
the
nanoplatform is modular, there are options of combinatorially incorporating
various different
ligands, pro-ligands, or immunomodulators to the nanoplatform. One unique
feature of the
immune-nanoplatform is that the nanofibrillar network formed at the tumour
microenvironment
is durable, which may explain its remarkable in vivo anti-tumour immune
response and memory
effects but without any sign of systemic immunotoxicity, even when given in
conjunction with
anti-PD-1 antibody. The pro-ligand concept of using LLP2A to capture T-cells
at the tumour site
is innovative, and may be applied for capturing other beneficial immune cells,
including natural
killer cells. Other potent immunomodulators against other pathways such as the
stimulator of
IFN genes (STING) pathway may also be tried. The nanoplatform is highly
modular and may
appear to be complicate. However, in reality, it is highly robust. Each
transformable peptide
.. monomer is chemically well-defined, and the final immune-nanoparticle can
be assembled by
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simple mixing in DMSO followed by dilution with water. Scale-up production for
clinical
development should not be a problem.
[0200] Statistical analysis. Data are presented as the mean standard
deviation (SD). The
comparison between groups was analyzed with the student's t-test (two-tailed).
The level of
significance was defined at *p < 0.05, **p < 0.01 and ***p < 0.001. All
statistical tests were
two-sided.
[0201] Although the foregoing invention has been described in some detail by
way of
illustration and example for purposes of clarity of understanding, one of
skill in the art will
appreciate that certain changes and modifications may be practiced within the
scope of the
appended claims. In addition, each reference provided herein is incorporated
by reference in its
entirety to the same extent as if each reference was individually incorporated
by reference.
Where a conflict exists between the instant application and a reference
provided herein, the
instant application shall dominate.
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SEQUENCE LISTINGS:
SEQ ID NO:1: KLVFF
SEQ ID NO:2: klvff
SEQ ID NO:3: FFVLK
SEQ ID NO:4: YCDGFYACYMDV
